Experiment 4: SPECTROPHOTOMETRY / COLORIMETRY

(Eda ALGÜL), Gebze Technical University, Turkey

SUMMARY
This experiment focuses on the application of spectrophotometry to analyze Methyl Red, a pH
indicator known for its distinct absorption properties. By measuring the absorption of light at
various wavelengths, we aim to establish its absorption spectrum and identify the wavelength
of maximum absorbance. Utilizing the Beer-Lambert Law, we will prepare a standard curve
that correlates absorbance with concentration, enabling us to determine the concentration of an
unknown Methyl Red sample. This practical investigation not only enhances our skills in using
a spectrophotometer but also deepens our understanding of the principles governing light
absorption in colored solutions, laying the groundwork for further analyses of similar
compounds in biochemical research.

INTRODUCTION molar absorptivity is ε, the concentration is

c, and the path length is l.
Spectrophotometry is a technique used to
measure the amount of light absorbed or
Methyl Red, a pH indicator commonly used
transmitted. reflected, and/or emitted by a
in titrations, exhibits distinct absorption
substance as a function of its wavelength,
properties that vary with pH, making it an
concentration of the sample, and path length
excellent candidate for spectrophotometric
or distance the light traverses through the
analysis. Its bright color and strong
substance.(1)This method involves
absorbance in the visible spectrum enable
measuring the absorption of light by a
accurate measurements, thereby facilitating
substance at different wavelengths,
the determination of its concentration in
allowing for the determination of
unknown samples. By first establishing the
concentration and other characteristics of
absorption spectrum of Methyl Red, we can
the analyte. In particular, Beer-Lambert law
identify the wavelength of maximum
describes the relationship between the
absorbance, which is crucial for optimizing
absorbance of light by a substance, the
assay sensitivity.
concentration of the substance, and the path
The objective of this experiment is twofold:
length of the light through the sample.(2) In
to determine the absorption spectrum of
spectrophotometry, this law plays a
Methyl Red under controlled conditions and
fundamental role in quantifying the
to prepare a standard curve that relates
concentration of a solute in a solution. The
absorbance to concentration. This standard
law is expressed as
curve will be instrumental in assessing the
concentration of an unknown Methyl Red
A = εcl
sample. Furthermore, the principles learned
from this experiment can be applied to
In the above expression, A refers to the
analyze other colored compounds,
absorbance. The extinction coefficient or
enhancing our understanding of
spectrophotometric techniques in RESULTS
biochemical assays. Through this
experiment, we aim to gain practical The absorbance measurements for each
experience in using a spectrophotometer prepared sample (s0-s5) showed a clear
and develop a comprehensive correlation between Methyl Red
understanding of the factors influencing concentration and absorbance values, with
light absorption in colored solutions. higher concentrations yielding greater
absorbance. The maximum absorbance
EXPERIMENTAL PROCEDURES wavelength was identified at approximately
[insert specific wavelength here] nm,
consistent with Methyl Red’s expected
A pipette was used to measure 1 mL of
absorption peak at pH 4. The unknown
Methyl Red solution (5 µL/mL). Then, 1
sample (s6) displayed an absorbance value
mL of 1 M sodium acetate buffer was added
falling between those of s3 and s4,
to the same container. This ensured that the
suggesting a concentration near 2 µg/mL.
pH of the solution remained at 4. The
Control wells showed negligible
cuvette was placed into the
absorbance, confirming the accuracy of the
spectrophotometer. The spectrophotometer
blank used. The results of the experiment
was set to scan between 400 nm and 600
are shown in table-1 and graph-1.
nm, using the pH 4 buffer as a blank control.
The absorption spectrum of Methyl Red
was recorded, and the wavelength at which Concentration average-
absorbance reached its maximum was Samples (µg/mL ) Abs1 Abs2 Average blank
determined. This wavelength represents the S0 5 0,815 0,828 0,8215 0,791
maximum absorption of Methyl Red at pH S1 4 0,65 0,695 0,6725 0,642
4. S2 3 0,487 0,488 0,4875 0,457
In the second phase of our experiment, S3 2 0,374 0,345 0,3595 0,329
preparations were made using six tubes, S4 1 0,232 0,203 0,2175 0,187
labeled s0, s1, s2, s3, s4, s5, and s6. The S5 0,2 0,117 0,077 0,097 0,0665
contents of the tubes are as follows: B 0,028 0,033 0,0305 0
Unknown 2,77 0,463 0,432 0,4475
- s0 = 2.5 mL of Methyl Red Table-1: The absorbance values according
- s1 = 2 mL of Methyl Red + 0.5 mL of to different concentrations of Methyl Red
buffer
- s2 = 1.5 mL of Methyl Red + 1 mL of
buffer Calibration curve
- s3 = 1 mL of Methyl Red + 1.5 mL of 1
y = 0,1505x + 0,0308
buffer R² = 0,9977
0,8
- s4 = 0.5 mL of Methyl Red + 2 mL of
Absorbance

buffer 0,6
- s5 = 0.1 mL of Methyl Red + 2.4 mL of
0,4
buffer
- s6 = unknown 0,2

0
These tubes were distributed into wells 0 1 2 3 4 5 6
using a pipette, each maintaining a total
Concentration (µg/mL)
volume of 2.5 mL. The remaining two wells
were filled with buffer solution as a control
group. The loaded wells were then placed Graph-1: Absorbance–Concentration
into the device to measure their absorbance. (µg/mL) graph
DISCUSSION unknown sample aligns well with the trend
observed in the prepared standards,
In this experiment, the absorbance of confirming the reliability of the technique
Methyl Red at various known when applied to Methyl Red.
concentrations was measured using a
spectrophotometer, enabling the generation REFERENCES
of a standard curve for quantitative analysis. 1) Beckman AO, Gallaway WS, Kaye W,
The concentrations of the samples ranged Ulrich WF. History of spectrophotometry at
from 0.2 µg/mL to 5 µg/mL, with the Beckman instruments, Inc. Analytical
absorbance readings obtained from chemistry. 1977 Mar 1;49(3):280A-300A.
duplicate measurements. These readings
were averaged to increase accuracy, and the 2) Delgado R. Misuse of Beer–Lambert
blank absorbance (0.0305) was subtracted Law and other calibration curves. Royal
from each average to yield corrected values Society open science. 2022 Feb
for each concentration. This step is essential 2;9(2):211103.
in spectrophotometric analyses to account
for any baseline absorbance that might
interfere with the accuracy of the results.

The results indicate a positive correlation

between the concentration of Methyl Red
and its absorbance, consistent with Beer-
Lambert Law, which states that absorbance
is directly proportional to the concentration
of the analyte under constant path length
and molar absorptivity. At the highest
concentration (5 µg/mL), the corrected
average absorbance was 0.791, and it
progressively decreased with lower
concentrations. For the unknown sample,
the measured absorbance was
approximately 0.4475 (average corrected
for blank), corresponding to a concentration
of around 2.77 µg/mL based on the standard
curve.

Minor discrepancies in duplicate

absorbance readings across samples may be
due to slight inconsistencies in pipetting,
small fluctuations in spectrophotometer
readings, or sample handling. These
variations are relatively minimal and do not
significantly impact the overall trend
observed in the data.

The experiment successfully demonstrated

the effectiveness of spectrophotometry in
determining the concentration of an
unknown sample by establishing a standard
curve. The calculated concentration for the