UNIVERSITY OF GHANA, LEGON

DEPARTMENT OF BIOCHEMISTRY, CELL AND MOLECULAR BIOLOGY

EXPERIMENT 9

USING SPECTROPHOTOMETER TO DETERMINE THE WAVELENGTH

OF MAXIMUM ABSORPTION AND BEER - LAMBERT’S LAW

JOSEPH ARMAH BAIDOO

ID: 10852150

SEPTEMBER 28, 2021

GROUP 15

MABEL APPIAH- ADJEI

KUUMAZAH BLESS KWAKU

ANAAFI- KWAPONG CHRISTIAN

MARFO SAMUEL

KWAYISI- DARKWAH CALEB KORANTENG

ISHAAQ AGBERE NAJMU- DEEN

OWUSU EMMANUEL DONKOR

JOSEPH BAIDOO
INTRODUCTION

Spectrophotometer is a device used to measure the amount of light absorbed or

transmitted by a sample. Spectrophotometric methods help measure the concentration

of solutes in a sample liquid ( a coloured sample) by measuring the amount of light

the sample absorbs ( Zumadahl & Zumadahl, 2007). Coloured samples are usually

used in spectrophotometry. This is because, coloured solutions have the ability to

absorb light at a very specific wavelength and hence, are able to transmit the colour

they absorb. This is how we are able to see the colours the possess (Barbour, 1929;

Klein et al., 2015). When a coloured solution is placed in a spectrophotometer, a light

source incidents on it. The light source first hits the grating which separates the white

light into its spectrum according to their wavelength and also allows the wavelength

to be varied. Once a desired wavelength has been set, the grating allows light with the

desired wavelength to incident on the sample in a cuvette. A detector then reads the

amount of light absorbed and transmitted by the sample. Transmittance refers to the

amount of light that completely passes through the sample while absorbance refers to

the measurement of light that is accumulated by the sample.

According to Van Der Vies (1953), spectrophotometer is a very important device

used in Clinical Chemistry, Molecular Biology, Immunology and Hematology. A lot

can be known from knowing how a sample relates with light. Many scientists have

studied the spectrophotometer and have come up with remarkable equations

concerning the principles underlying the use. One of such equations is the Beer -

Lambert’s law. Beer - Lambert’s law is formulated by two scientists; Johann Lambert

and August Beer. It relates the absorbance of light by a sample in the

spectrophotometer to the concentration of solutes in the sample; A= Ꜫx C x L. Beer -

Lambert’s law implies that the more concentrated the solute is in the sample, the more
light the sample will absorb. Hence a highly concentrated solution will absorb more

light at a particular wavelength than a less concentrated solution ( Cox et al, 2000).

SPECIFIC OBJECTIVE: This experiment sought to determine the wavelength of

highest absorption of three samples and also to determine Beer - Lambert’s law.
MATERIALS

UV Spectrophotometer, Methylene blue (100 mg/L), Methyl orange (100 mg/L) and a

sample of an unknown mixture.

METHOD

The experiment was in two parts. The first part was to determine the wavelength of

light at which the three samples given would record their respective highest

absorption.

In the first part, a stock solution of 100 mg/L of each sample was given. Each solution

was diluted to 10 mg/L and the rest were kept. The new stock solutions were now sent

to the spectrophotometer for recording. The spectrophotometer was first zeroed using

distilled water as a blank. Each sample was accounted and recorded for according to

the data given by the spectrophotometer. The absorbance of each samples were

determined at the following wavelengths: 400, 420, 440, 460, 470, 480, 490, 520, 540,

565, 580, 620, 660 and 700 all in nm. The spectrophotometer was zeroed with the

distilled water as a blank, each time a sample had to be changed or reread. Each

absorbance value, for all three samples, at all wavelengths was recorded twice and the

average was determined. This was done to ensure accuracy and precision. A graph of

absorbance against wavelength was plotted for this experiment.

In the next part of the experiment, Beer - Lambert’s law was to be determined using

the results in part A. In this experiment, six test tubes were set apart each for only two

of the samples; methylene blue and the methyl orange.Using the same solutions as in

part A, a mixture of each sample and water were obtained according to the ratio
given in table one below:

TABLE 1:THE VOLUME OF INDICATORS USED IN THE BEER - LAMBERT’S

EXPERIMENT

Test tube No 1 2 3 4 5 6

Volume of indicator 0 1 2 3 4 5
(ml)

Volume of distilled 5 4 3 2 1 0
water (ml)

TABLE 1: SHOWS THE VOLUME OF DISTILLED WATER AND VOLUME OF

SAMPLE USED IN THE SECOND EXPERIMENT.

After adding the volumes of sample and distilled water according to the Table 1, the

resulting mixture were inserted into the spectrophotometer and the absorbance for

each sample were recorded twice. The average was taken. At each test, when the

sample was removed, the spectrophotometer was zeroed. Distilled water was used as

the blank. Each reading was taken at the highest wavelength of maximum absorption

for each sample. The highest wavelengths of maximum absorption for each sample

were determined in part A of the experiment. A graph of absorbance against

concentration was drawn for each sample and the data were used to determine the

concentrations of each solutions.

RESULTS

The absorbance of methylene blue, methyl orange and the unknown sample were

measured at specific wavelengths. This was done to determine the wavelength at

which the samples will record their respective highest absorption. For methylene blue,

the highest wavelength for maximum absorption was recorded at a wavelength of 620

nm. The absorption at this wavelength was 0.867. The highest wavelength for

maximum absorption determined for the methyl orange was recorded at 460 nm and

the absorption was 1.040. The unknown sample, however, produced two wavelengths

at which it records its highest absorption. The wavelengths were 460 nm and 470 nm.

The absorption recorded at these two wavelength was 0.298.

With the absorption values determined with the help of the spectrophotometer, a

graph of absorbance against wavelength was drawn to represent the information

recorded. A curve was obtained.

Figure 1. A graph of absorbance against wavelength of the three samples

Methylene Blue(Average)
1.2
Methyl Orange (Average)
Unknown(Average)
1

0.8

0.6

0.4

0.2

0
400 420 440 460 470 480 490 520 540 565 580 620 660 700

In figure 1 above, the graph shows the peak (wavelength of maximum absorption) of
the three samples. The methyl orange reaches the highest peak, followed by the

methylene blue ans finally the unknown mixture.

Figure 2. A graph of absorbance against concentration of methyl orange indicator at

its highest wavelength of maximum absorption

A table of concentration(mg/L) against average

absorption in methyl orange
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 2 4 6 8 10 12
Figure 3. A graph of absorbance against concentration of methylene blue at its highest

wavelength of maximum absorption.

A table of concentration(mg/L) against average

absorption in methyl orange
0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0
0 2 4 6 8 10 12
DISCUSSION