Chem.

Educator 2012, 17, 163–165 163

Methyl Red: A Chameleon That Reveals The Partition Between Two

Phases As A Function Of pH. An Extraction Experiment

M. Jazmín Silvero, Fabio E. Malanca, Cesar G. Gómez, Juan C. Fraire and Gerardo A.
Argüello*

INFIQC – Facultad de Ciencias Químicas – Universidad Nacional de Córdoba – Ciudad Universitaria – 5000
Córdoba, ARGENTINA, gerardoa@fcq.unc.edu.ar
Received April 26, 2012. Accepted July 14, 2012.

Abstract: “A picture is worth a thousand words,” and besides it is easier to remember. This work is based on that
premise, and its main objective is to show that creating a strong visual impact on the student’s mind is a useful
tool to teach important chemistry concepts. The activities proposed involve a biphasic medium composed of an
aqueous solution at different pH levels and petroleum ether (60–80C), where an acid-basic indicator such as
methyl red is employed to examine the phenomenon occurring in each phase, as it changes its color like a
“chameleon.” During the process, students are introduced to core concepts of physical chemistry through a
solvent partition system. The experiment is suitable for both qualitative and quantitative analyses of the
extraction process.

Introduction Methyl red shows the presence of several conformers in

equilibrium in the organic phase. Mukherjee et al., who studied
Extraction is a procedure widely employed in laboratories as the UV-Vis absorption of methyl red in different solvents,
well as in industries to separate the components of a mixture. demonstrated that the absorption intensity is mostly provided
In particular, this process can be used to either isolate or purify by quinoid zwitterions (Scheme 1) [4]. Moreover, in methyl
a component. Considering its relevance, it is important to make cyclohexane they observed the presence of maxima at 475 nm
students aware of the physical-chemical foundations of the and 455 nm corresponding to two zwitterions, and another
partition phenomenon. maximum at 420 nm belonging to a trans conformer. A similar
Various methods were used to introduce concepts such as spectrum was observed in our assays when petroleum ether
partition of a solute between two immiscible layers, was used as solvent (Figure 1), probably as a consequence of
Distribution Ratio (D), and Distribution Coefficient (KD) [1– the presence of the same species proposed by Murkherjee. The
3]. These experiments were designed to demonstrate the species concentration ratio depends both on the solvent and on
principles of separation of acid, basic, and neutral species in the temperature, but it was univocally determined when these
liquid-liquid extraction, and also the construction and use of a parameters did not change.
Lambert-Beer standard curve. However colorless species were HMR Isomers CH3
often used, and tabulated pKa were employed. In a previous N
N N
CH3
experiment, Sonnenberger and Ferroni used methyl violet C O
H
O
(which under their experimental conditions had the same color N N
CH3 O C O
N N
CH3

in both the organic and the aqueous phase) to determine KD. N

H
CH3 N
H
CH3

However, the pH influence on the equilibria involved in the C O

extraction procedure and the determination of D and Ka, were
not objects of study. In our case, both the visualization of all
the colored species in equilibrium and the determination of all
the constants involved are combined in a single experiment.
Selection of the solute is not an easy step, particularly if
additional conditions are required, namely that the species to
be extracted can be visualized in both phases, and that
variation in the solute concentration can be determined on the
basis of changes in color intensity. The chosen solute was
methyl red (HMR), 2-[4-(dimethylamino)phenylazo]benzoic
acid, a pH indicator whose acid form is extracted by petroleum
ether, yielding a yellow organic solution sharply contrasting
with the red aqueous phase observed at the same pH value, a
feature that allows students to easily separate the two layers.
This compound also changes from red to yellow with an
increase of the pH given by the variation in the acid-basic
species ratio in the aqueous phase.
O
Scheme 1. Equilibria present in the organic phase.
In an aqueous solution methyl red is a zwitterion with two
resonance structures (Scheme 2). The acid form (HMR) is
present mainly at a pH lower than 4.1, coloring the aqueous
phase red, and the basic form (MR–) at a pH higher than 6.1
turns the aqueous phase yellow. This behavior is associated
with the pKa value and the acid-basic equilibrium of the
indicator in an aqueous solution. A pKa value of 5.00 at a
temperature of 20 °C is reported in the literature [5].
In the present work, we propose the use of the methyl red
indicator as a solute capable of displaying the partition and its
pH dependence. This visual phenomenon is suitable for a
qualitative understanding. In addition, a quantitative analysis
of the solute allowed determination of D, KD and Ka (acid-base

© 2012 The Chemical Educator, S1430-4171(12)12437-8, Published 08/31/2012, 10.1333/s00897122437a, 17120163.pdf

164 Chem. Educator, Vol. 17, 2012 Argüello et al.

and after Aorg the extraction respectively at a given pH, using

Organic phase
HMR
the following equation:
KD

Aorg
D (1)

Aqueose phase
O O O
ini
C O

N
N N
CH3

CH3
C O

N
N N
CH3

CH3
Ka
C O

N
N N
CH3

CH3
+ H
Aorg  Aorg
H H

HMR MR-

This equation can be used because:

Scheme 2. Distribution of methyl red between the aqueous
solution and the organic phase.  solute in all its forms org
D
0.8
 solute in all its forms aq
max=454 nm

0.6
and
Absorbance

0.4
 soluteaq   soluteini
org 
 solute org

0.2 then,

0.0  soluteorg
350 400 450 500 550 D
Wavenumber (nm)  soluteini
org 
 soluteorg

Figure 1. Spectrum of methyl red in petroleum ether. according to the absorption spectra (Figure 1), the maximum
observed at 454 nm should correspond to one of the
equilibrium species present in the organic phase, and not to the
dissociation constant of the indicator) by measuring the analytical concentration of the solute. However, at this
absorbance in the organic phase via a colorimetric method. wavelength the colorimetric analysis becomes valid as D is a
concentration ratio. Therefore, all the common constant factors
Experimental are cancelled.
On the other hand, considering the relationship between D,
The experimental setup consisted of 10 groups of 2 students each KD, and Ka [5, 6]:
one, in a regular four-hour laboratory class.
Reagents & Equipment. The experiment required the use of the
following elements: vortex, visible spectrometer, glass cell, Pasteur K D  H 2O  
D (2)
pipette, test tubes, a solution of methyl red in petroleum ether 60–80  H 2O    K a
(Note 1), and aqueous solutions at different pH values (4.0, 4.5, 5.0,  
5.5 and 6.0) (Note 2).
Procedure. Place the organic solution in a glass cell, and measure and rearranging eq. 2, eq. 3 is obtained:
ini
the absorbance at 454 nm Aorg ).

1  H 2O   K a
Prepare five test tubes: using a pipette, place in each one 4.0 mL of
a different solution whose pH is in the 4.0-6.0 range.  (3)
Using a pipette, add 4.0 mL of the methyl red solution to each test D K D  H 2O  
tube. Shake the mixture properly using the vortex (be careful to avoid
leakage), and let it stand still so that the phases separate completely
and the two layers can be clearly observed. The organic phase which can be expressed in the form y = ax + b:
corresponds to the upper one because its density is lower than the one
of the aqueous solution.
1 Ka 1 1
Extract the organic phase from each test tube with a Pasteur pipette   (4)
and measure the absorbance at 454 nm using a glass cuvette. D K D H 2O  K D
Calculate the values of D and plot 1/D as a function of 1/ [H3O+].
Values of KD and Ka can be obtained from this plot as explained
From a plot of 1/D vs. 1/[H3O+], and can be obtained using
below.
the values of the slope and the y-axis intercept.
Figure 2 exhibits the results of a typical experiment that
Discussion reveals the dependence of D of methyl red on the pH of the
D, KD and Ka determination. According to the Lambert– solution. A more quantitative analysis showing the variation of
Beer law, A = ε b c, where ε is the molar absorptivity (at a 1/D with pH is provided in Figure 3 and Table 1, from which
given wavelengh), b the optical path, and c the analyte the values for KD = 8.2 and pKa = 5.09 are obtained, the latter
concentration, the Distribution Ratio (D) can be determined by being in agreement with that reported in the Handbook of
ini
Chemistry [6].
measuring the absorbance of the organic phase before Aorg

© 2012 The Chemical Educator, S1430-4171(12)12437-8, Published 08/31/2012, 10.1333/s00897122437a, 17120163.pdf

Methyl Red: A Chameleon That Reveals The Partition... Chem. Educator, Vol. 17, 2012 165

Table 1. Values of D obtained from absorbance data at different pH observed, it is possible to discuss with the teachers the
Abs pH +
(1/[H3O] ) × 10 –4
D 1/D optimum pH range to perform an efficient extraction.
0.324 4.00 1.00 7.81 0.13
0.319 4.50 3.16 6.89 0.15 Caution
0.300 5.00 10.0 5.14 0.22
Eye protection, gloves, and a fume hood must be used. This
0.253 5.50 31.6 2.27 0.44
experiment employs petroleum ether, a flammable liquid with
0.175 6.00 100 0.92 1.10
moderate vapor pressure (direct contact with the flame must be
avoided). It is harmful if swallowed or inhaled. It causes skin
and eyes irritation and it also may affect the central nervous
system. The flammable residuals should be properly disposed
of. During the whole experiment gloves must be worn for the
handling of ether, and acid and basic solutions.

Notes

A solution of methyl red was previously prepared using

petroleum ether 60-80 as solvent. A simple procedure to
prepare 500mL of saturated solution was followed by adding
Figure 2. Test tubes containing both organic (upper) and aqueous petroleum ether to 0.020 g of the dye and stirring for about 3.0
(lower) phase at a selected pH (4.0, 4.5, 5.0. 5.5 and 6.0). h. The resulting solution was filtered before storage. Its
absorbance at 454 nm was ranged between 0.5 and 0.7.
Aqueous buffer solutions of PBS (100 mL of 100 mM
1,2 Phosphate Buffer Solution) were prepared for each pH. The
pH was adjusted between 4.0 and 6.0 (step 0.5) by using a pH-
meter and by the adding either NaOH or HCl.
The volumes of aqueous and organic layer need to be strictly
0,8
the same. Therefore, the volume of both the organic and the
aqueous layer should be measured accurately.
1/D

0,4 Acknowledgements. M. Jazmín Silvero wants to specially

thanks her parents. We gratefully acknowledge the School of
Chemical Sciences (National University of Córdoba) for
0,0
supports. We would also like to thank Marisa Paz, Adrián Di
0,0 0,4 0,8 1,2 Fiore, and Fernanda Campestrini for their assistance with the
+
1/[H3O ] x 10
6 setting, and all the students who contributed their suggestions
to improve the activities presented.

Figure 3. Plot 1/D vs. 1/ [H3O+]. The values of Ka and KD were References and Notes
determined from the ratio of the slope and the y-axis intercept.
1.
Conclusions
2.
The activity described above takes about four hours in a 3.
chemistry laboratory, and it is possible to conduct either a
qualitative (in a shorter time) or a quantitative analysis: the 4.
former, through the comparison of the color solutions, the
latter by means of a visible spectrometer. The experiment was 5.
performed as a practical assignment for a second-year
undergraduate course at the School of Chemical Sciences
(National University of Córdoba) during the last five years. 6.
The satisfactory evaluation of more than 2,500 students reveals
that they grasped the basic concepts and procedures involved 7.
in the task discussed.
In a quantitative analysis this experiment allows to
determine the values of D and KD of methyl red between
aqueous solution and petroleum ether (60-80), as well as its
acid dissociation constant (Ka). According to the data
Sonnenberger, D. C. and. Ferroni E. L, J. Chem. Educ. 1989, 66(1),
91–92.
Turner, D. E., J. Chem. Edu. 1994 71(2), 173–174.
Kealey, D.; Haines, P. J.; Instant Notes – Analytical Chemistry BIOS
Scientific Publishers Limited, 2005, England, pp 109–118.
Mukherjee S. and Bera S. C., J. Chem. Soc., Faraday Trans., 1998,
94, 67–71.
Lide, D. R., CRC Handbook Chemistry Physics 85, Editor-in-Chief,
Former Director, Standard Reference Data, National Institute of
Standards and Technology.
Day, R. A.; Jr; Underwood, A. L. Química Analítica Cuantitativa,
5a. edición, España, 1989, pp. 556.
Burriel Martí, F.; Lucena Conde, F.; Arribas Jimeno, S.; Hernández
Méndez, J. Química Analítica Cualitativa, decimoctava edición,
España, 2001; pp. 296.

© 2012 The Chemical Educator, S1430-4171(12)12437-8, Published 08/31/2012, 10.1333/s00897122437a, 17120163.pdf