Exercise No.

1
UV- VISIBLE SPECTROPHOTOMETRY

Gerry Mark Gubantes

CHEM 137.1 – 2L
2nd Semester AY 2017-2018

Date Performed: February 3, 2018

Date Submitted: February 9, 2018

Mr. Jethro Magsangkay

Laboratory Instructor
 Abstract
In the experiment, the linear relationship between absorbance and
concentration at a wavelength and deviations from the Beer’s Law
Plot were illustrated. The maximum wavelengths were determined
at 512, 394 and 529 nm for Co, Ni and KMnO 4, respectively. The
concentration of an unknown sampled of Co and Ni solutions were
0.01756 (1030.656 mg/L) and 0.07795 (4593.84 mg/L) M,
respectively. Lastly, spectrophotometric analysis of a multi-
component system was done. It was used to calculate the
concentration of benzoic acid and caffeine in the mixture which
were found to be 1.6603x10-3 and 1.2320x10-3 M, respectively.

I. Introduction

The Ultraviolet-visible (UV-Vis) spectrophotometry refers to the absorption or reflectance

spectroscopy in the ultraviolet-visible spectral region. This region, moreover, is divided into three
sub-domains termed near UV (185–400 nm), visible (400–700 nm) and very near infrared (700–
1100 nm). Most commercial spectrophotometers cover the spectral range of 185 to 900 nm
(Rouessac and Rouessac, 2007). This method uses the instrument UV-Vis spectrophotometer.
Generally, the instrument has five basic components namely light source, sample holder,
wavelength selector, detector or transducer, and signal processor and readout (Figure 1.1).
The light source is a stable source of radiant energy. It can be either continuous or
discontinuous source. Continuous sources such as tungsten and deuterium lamp produce
spectra over a broad range. On the other hand, discontinuous or discrete sources such as
hollow cathode lamp (HCL) and electrodeless discharge lamp (EDL) produce only at specific
wavelengths. Furthermore, the sample holder is a transparent container which holds the
sample. Cuvette, which is commonly available in 1, 5, & 10 cm path lengths, is the most widely
used sample holder. It can be a glass, plastic, quartz or fused silica. The wavelength selector,
moreover, is a device that isolates a restricted region of the spectrum for measurement. It can
be an optical filter or monochromator. Likewise, the detector or transducer converts radiant
energy to a usable signal. The commonly used detector are photovoltaic cells, phototubes,
photomultiplier tubes (PMT’s), photoconductivity and silicon photodiodes. Lastly, the signal
processor and readout display the transduced signal. It can in the form of computer display,
digital or analog readout, strip chart recorder and integrator (Skoog, et al., 1998).
The UV-Vis spectrophotometer measures the intensity of light passing through a sample
(P), and compares it to the intensity of light before it passes through the sample (Po). Getting the
 P
ratio Po will give the transmittance which is usually expressed as a percentage (%T). The
absorbance is then related to transmittance given by the equation below (Skoog, et al., 1998).
Po 1
Abs=log( )=log ( )=−log T =abc
P T (1.1)
The spectrum of the compound under analysis will be obtained as a graph representing
the transmittance (or the absorbance) as a function of wavelength along the x- axis, usually
given in nanometers. Moreover, all the absorption analyses are governed by the Beer-Lambert’s
law (Equation 1.1). The Beer’s plot can be prepared by measuring the light absorbed by the
solutions of varying concentration. Using the relationship between the two, Equation 1.1 can be
represented as (Skoog, et al., 1998).:
Abs=εbc (1.2)
Two compounds in a solution, for example, when analyzed will give different absorbance
maxima (λmax). This will enable their simultaneous determination in the solution. Since the
absorbance (Abs) of a solution containing both compounds at particular wavelength are
additive, the concentration (c) of each compound can be determined by choosing two
wavelengths (λ1 and λ2) on which to observe the Abs.
Abs λ 1=ε λ 1 bc 1 +ε λ 1 bc 2
Abs λ 2 =ε λ 2 bc1 +ε λ 2 bc 2 (1.3)
The general objective of this experiment is to employ UV-Vis spectrophotometry
instrumentation. Specifically, it aims (1) to perform basic procedures in evaluating condition and
performance of a UV-Visible spectrophotometer, (2) to determine and compare the absorption
spectrum of the various solutions, (3) to illustrate the linear relationship between absorbance
and concentration at a particular wavelength, (4) to illustrate deviations from Beer’s Law plot, (5)
to experience the use of a double beam spectrophotometer and/or a scanning UV-visible
spectrophotometer, (6) to familiarize the spectrophotometric analysis of a multi-component
system, and (7) to determine the concentrations of unknown substances.
 II. Methodology

Initially, the instrument was calibrated to check if it is operating properly. The instrument
used in this experiment was UV Mini-1240 Spectrophotometer, which is a single beam
spectrophotometer (Figure 1.2).

Figure 1.1. UV Mini-1240 Spectrophotometer, a single beam spectrophotometer.

For the absorption spectrum and Beer-Lambert’s Law of Linearity, the following steps
were followed. First, the instrument was turned on and allowed to warm up for 30 minutes.
Then, 25 mL of 0.05 M, 0.10 M, 0.15 M, and 0.20 M solutions were prepared using the 0.50 M
stock solution of Co, Ni and KMnO4. After that, the absorbance of the solution with highest
concentration, 0.20 M, was read from 700 to 380 nm. The absorbance against wavelength was
plotted to determine the wavelength of maximum absorption, λ max. Then, the absorbance of the
solutions and unknown was determined at the determined λmax.
For the simultaneous analysis of two-component system, benzoic acid and caffeine were
used. Fifty milliliters of each standard solutions were prepared. The concentrations of benzoic
acid solutions were 2.00 mg/L, 4.00 mg/L and 8.00 mg/L. On the other hand, the concentration
of caffeine solutions were 4.00 mg/L, 8.00 mg/L and 16.00 mg/L. The required amount of stock
solution was obtained, followed by the addition of 0.10M HCl and dilution with distilled water.
Also, 50.0 mL of a mixture of 4.00 mg/L benzoic acid and 8.00 mg/L caffeine was prepared. The
absorption spectrum of 4.00 mg/L benzoic acid solution, 8.00 mg/L caffeine solution, and the
benzoic acid-caffeine solution was determined from 350 to 210 nm. Using the obtained spectra,
two appropriate wavelengths for a Beer’s Law plot was chosen.
Lastly, the concentrations of benzoic acid and caffeine in three different soft drink sample
were determined. The sample used was Sprite. Twenty milliliters of each sample were heated in
a beaker to expel CO2. Any particles were removed via filtration before it was cooled in room
temperature. A 0.50 mL of each sample were placed in a 25 mL volumetric flask. Before it was
diluted with dH2O up to mark, 0.10 mL HCl solution was added. Instead of using the sample,
dH2O was used as method blank. The %T or Abs was read and recorded. Using the calibration
curve, the concentrations of benzoic acid and caffeine were calculated.

III. Results and Discussion

The colors of coordination compounds are complementary in nature. If light passed

through a substance and absorbs the color red, for example, the color that can be observed
would be green, since it is the complementary color of orange. The color wheel illustrates the
complementary colors (Skoog et al., 1998).

Figure 1.2. The color wheel.

The amount of light absorbed by a substance can be measured in terms of transmittance
and absorbance. Absorption occurs when light passes through an absorbing sample, and the
absorbing species absorb some of the light while transmitting the rest. Incident radiation, Po,
enters the sample which then absorbs some of the radiation and transmits others; this radiation
can be labeled as P. The transmittance is then measure by taking the ratio of the difference in
amount of light (Skoog et al., 1998).
Spectrophotometers are spectrometers that allow measurement of the ratio of the
radiant powers of two beams, a requirement to measure absorbance. They can be classified
either as double-beam or single beam instrument.
 Figure 1.3.a. Block diagram of a single-beam spectrophotometer.

Figure 1.3.b. Block diagram of a double-beam spectrophotometer.

In single-beam instruments, measuring the transmittance of the sample and of the
solvent at each wavelength is required to obtain a spectrum. A control corresponding to the
solvent alone or a solution containing the reagents of the measurement is placed in the optical
path. Then, it is replaced by the solution prepared from the sample of unknown concentration. It
should be noted that it does not contain the compound to be measured. Its advantages include
the price and ruggedness. However, single beam instrument is not practical for recording
spectra since manually adjusting the wavelength and recalibrating the spectrophotometer is
tedious and time-consuming. Also, the accuracy of a single-beam spectrophotometer is limited
by the stability of its source and detector over time (Rouessac and Rouessac, 2007; Harvey,
2000).
In double beam instrument, the light source can be passed (simultaneously) through
both a reference and a sample cell. When the light passed through, it will be splitted into two
separate beams through the aid of the chopper. Moreover, the light output of the instrument is
adjusted to 100% transmission (0 % absorbance). This instrument allows the correction of the
sample absorbance signal for non-analyte absorbance (Rouessac and Rouessac, 2007).
In setting these spectrophotometers at some particular wavelengths, light is emitted in
Gaussian light distribution as shown below. The spectral bandwidth is the width of the band of
light at one-half the peak maximum. It is related to the physical slit-width of the monochromator
which in turn affects the resolution capabilities of the instrument. Resolution is the ability of an
instrument to separate light into finite, distinct wavelength regions and distinguish these finite
regions from each other. The resolution and the sensitivity of the instrument is at maximum at
the λmax where the light intensity is at peak (Allen, 2008).

Figure 1.4. Gaussian Distribution of Light Intensity.

For the absorption spectrum and Beer-Lambert’s Law of Linearity, 0.05 M, 0.10 M, 0.15
M, and 0.20 M solutions were prepared using the 0.50 M stock solutions of Co, Ni and KMnO 4.
The absorbance of the solution with highest concentration was read from 700 to 380 nm. This
was done to determine wavelength of maximum absorption, λmax. After obtaining the λmax, the
absorbances of different solutions were determined.

Table 1.1. The determination of the unknowns and the construction of the standard curve.
 Cobalt Standard
1
0.9
0.8 R²
f(x)==1 4.5x
0.7
Abs orbance

0.6
0.5
0.4
0.3
0.2
0.1
0
0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 0.22
Concentrati on

Figure 1.5. The standard curve for the solutions of Cobalt

Nickel Standard
0.3
0.25
f(x) = 1.24x + 0
Abs orbance

0.2 R² = 1
0.15
0.1
0.05
0
0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2 0.22
Concentrati on
As shown in Figure 1.5., the
standard curve shows a linear relationship between the cobalt and the absorbance. The
calculated values for the R2 and the slope (m) are 0.9994 and 4.49866, respectively. This R 2
value implies that the relationship between the glucose concentration and the absorbance for
the standard is linear enough to be utilized as a standard. The equation of the line was obtained as
y = 4.498666667x. Thus, the calculated concentration for the unknown sample of cobalt is 0.01756M
(1030.656 mg/L) with a percent error of 3.0656%.

Figure 1.6. The standard curve for Nickel.

As shown in Figure 1.6., the standard curve shows a linear relationship between the
nickel concentration and the absorbance. The calculated values for the R2, slope (m) and the y-
intercept are 0.9992, 1.2380 and 0.004500, respectively. This R2 value implies that the
relationship between the nickel concentration and the absorbance for the standard is linear
enough to be utilized as a standard. The equation of the line was obtained as y = 1.238000x +
0.004500. Thus, the calculated concentration for the unknown sample of nickel is 0.07795M
(4593.84 mg/L) with a percent error of 45,838%.

KMnO4 Standard
4.02
4 f(x) = 3.45x + 3.95
3.98 R² = 0.42
Abs orbance

3.96
3.94
3.92
3.9
3.88
3.86
0 0.01 0.01 0.02 0.02 0.03
Concentration
 Figure 1.7. The standard curve for permanganate.
Meanwhile, for the KMnO4 solution, the standard curve shows a not linear relationship
between the permanganate concentration and the absorbance. The calculated values for the R 2,
slope (m) and the y-intercept are 0.4224, 3.4455 and 3.9472, respectively. This R 2 value implies
that the relationship between the permanganate concentration and the absorbance for the
standard is not linear enough to be utilized as a standard. The equation of the line was obtained
as y = 3.445545x + 3.947240. No unknown sample for the KMnO4.
The different assumptions of this law are: (1) the incident radiation is monochromatic; (2)
the absorbing species act independently of each other in the absorption process; (3) the
absorption occurs in a volume of uniform cross-section; (4) energy degradation is rapid; and (5)
the refractive index is independent of concentration (Skoog et al., 1998).
However, Beer’s Law has limitations. First, it is valid only for low concentrations (≤0.01
M) of analyte. At higher concentrations, the electrostatic interactions between particles of
analyte may change the value of ε. Also, chemical deviations occur when the absorbing species
undergoes association, dissociation, or reaction with the solvent to give products that absorb
differently from the analyte. Added to these are the instrumental deviations such as
polychromatic radiation and stray light. For example, a polychromatic light reaches the sample
cell. This may result to either a negative or a positive deviation. The spectral band width of the
instrument has therefore a great effect on the quality of the light that passes through the
sample. A high spectral band width will lead to a less accurate absorbance measurement. Stray
light, on the other hand, will lead to a decrease in the absorbance of the sample since the
detector will detect a greater amount of light that is transmitted (Harvey, 2000).
Furthermore, the slope of the Beer’s law plot can be determined even if the only
available data is absorption spectrum and the knowledge that it obeys Beer’s law. Given that the
Beer’s law the plot is linear, the x value corresponds to the concentration of the solution while
the y value corresponds to the absorbance, the slope will be equal to y divided by x. Since the
path length is constant, the slope will be equal to the absorptivity (Harvey, 2000).
The obtained maximum wavelength of Co, Ni and KMnO4 were 512 nm, 394 nm and 529
nm. This means that at these wavelengths, all the colors of the spectrum are absorbed except
the colors that are emitted in each corresponding solution (red for Co, green for Ni and Dark
Violet for permanganate). These corresponding wavelengths would be desirable for the
analyses of solutions with concentrations 0.15 Co and 0.55 M Co because there is maximum
absorption at these wavelengths. This is also where the resolution and the sensitivity are at the
highest (Allen, 2008; Harvey, 2000).

The last part of the experiment involved the simultaneous analysis of two-component
systems. The absorbance of the prepared benzoic acid and caffeine standards were measured
at 273 nm and 230 nm. These wavelengths are the wavelengths where the solutions absorb
most.

Table 1.2. Summary of measurements for the %T and Abs of benzoic acid
Sample Concentration λ=273 nm λ=230 nm
Abs Abs
, mg/mL
benzoic acid 0 0 0
2 0.051 0.207
4 0.083 0.419
8 0.152 0.861
Caffeine 0 0 0
4 0.312 0.071
8 0.526 0.219
16 1.052 0.598
Blank --- 0.011441043 0.022276395
Mixture --- 0.575 0.695
 Absorbance at 273 nm
1.2

1 f(x) = 0.06x + 0.02

R² = 1
Concentration

0.8
Caffeine
0.6 Linear (Caffeine)
Benzoic Acid
0.4 Linear (Benzoic Acid)

0.2
f(x) = 0.02x + 0.01
0 R² = 0.99
0 2 4 6 8 10 12 14 16 18
Absorbance

Figure 1.8. Plot of absorbance vs concentration at 273nm for caffeine and benzoic acid.

Absorbance at 231 nm
1
0.9
0.8 f(x) = 0.11x - 0.01
0.7 R² = 1
Abs orbance

0.6 Caffeine
0.5 f(x) = 0.04x - 0.05 Linear (Caffeine)
0.4 R² = 0.97 Benzoic Acid
Linear (Benzoic Acid)
0.3
0.2
0.1
0
0 2 4 6 8 10 12 14 16 18
Concentration
Figure 1.9. Plot of absorbance vs concentration at 231 nm for caffeine and benzoic acid.

Table 1.3. Data for the analysis of the concentration of benzoic acid-caffeine solution.
ε (L mol-1 cm-1)
at 273 nm at 230 nm
Benzoic acid 0.0185 0.1078
Caffeine 0.0647 0.0385

From the data given, the two-systems of equation can now be constructed as follows:
Abs λ 1=ε λ 1 bc 1 +ε λ 1 bc 2
Abs λ 2 =ε λ 2 bc1 +ε λ 2 bc 2
0.2697 = (0.0185 L mg cm-1)(1cm)c1 + (0.0647 L mg-1 cm-1)(1cm)c2
-1

0.553= (0.1078 L mg-1 cm-1)(1cm)c1 + (0.0385 L mg-1 cm-1)(1cm)c2

The concentration of benzoic acid (C1) in the sample is determined as 202.7625 mg/L
(1.6603x10-3 M). Meanwhile, the concentration of caffeine (C2) in the sample is determined as
150.445 mg/L (1.2320x10-3 M) with a percent error of -1.376%. The literature value for caffeine
in mountain dew is 152.5445 mg/L (ABA, 2015).
The possible sources of errors in this experiment are the inclusion of possible contaminants,
the improper reading of the absorbance which can cause contaminations and the non-removal
of particles in the benzoic acid-caffeine sample.

IV. Sample Calculations

Determination of the Unknown concentration
absorbance−( y −intercept ) 0.079
Concentration= = =0.01756 M
slope 4.4987

Percent Error%
actual−theoretical 10 30.656−1000
%error= x 100= x 100=3.0656
theoretical 1000
Concentration of benzoic acid and caffeine

Using Equation 1.3,

Abs λ 1=ε λ 1 bc 1 +ε λ 1 bc 2
Abs λ 2 =ε λ 2 bc1 +ε λ 2 bc 2
0.2697 = (0.0185 L mg-1 cm-1)(1cm)c1 + (0.0647 L mg-1 cm-1)(1cm)c2
0.553= (0.1078 L mg-1 cm-1)(1cm)c1 + (0.0385 L mg-1 cm-1)(1cm)c2

c 1=[ benzoic acid ] =( 3.6454L mg )( 10001 gmg ) 122.12

1 mol
g
¿ x 50=1.6603 x 10−3
M

3.0089mg 1g 1mol
c =[ caffeine ] =(
2 ) ( ) −3
¿ x 50=1.2320 x 10 M
L 1000 mg 194.19 g

V. Conclusions

In the experiment, the absorption spectrum and Beer-Lambert’s Law of linearity were
studied using Co, Ni and KMnO 4 solutions. The linear relationship between absorbance and
concentration at a wavelength was illustrated and deviations from Beer’s law plot were also
determined. Using the absorption wavelength, the maximum wavelength of Co, Ni and KMnO 4 is
512, 394 and 529 nm, respectively. These wavelengths were used to determine Beer’s Law plot
for Co solutions that was further used to determine the concentration of the unknown solutions.
The concentration of the unknown solutions for Co and Ni were 0.01756 and 0.07795 M,
respectively.
Lastly, spectrophotometric analysis of a multi-component system was done to calculate
the concentration of benzoic acid and caffeine in the mixture. The molar absorptivities of both
compounds were obtained from the Beer’s Law plot. For benzoic acid, ε = 0.0185 (at 273 nm)
and 0.1078 (at 230 nm) while for caffeine ε = 0.0647 (at 273 nm) and 0.0385 (at 230 nm). Using
these parameters, the concentrations were calculated as 1.6603x10-3 and 1.2320x10-3 M for
benzoic acid and caffeine, respectively.

VI. References

AMERICAN BREWERY ASSOCIATION (ABA). 2015. Mountain Dew. Retrieved from

http://www.mountaindew.com

ALLEN, D. 2008. Understanding Spectral Bandwidth and Resolution in the Regulated

Laboratory.
New York: Thermoscientific Fischer Co.

HARVEY D. 2000. Modern Analytical Chemistry. 1st ed. New York: McGraw-Hill.

ROUESSAC F, ROUESSAC A. 2007. Chemical Analysis: Modern Instrumentation Methods and

Techniques. 2nd ed. Wiley.

SKOOG DA, HOLLER FJ, NIEMAN TA. 1998. Principles of Instrumental Analysis. 2nd ed.