Laboratory Experiment

pubs.acs.org/jchemeduc

Determination of Titratable Acidity in Wine Using Potentiometric,

Conductometric, and Photometric Methods
Dietrich A. Volmer,‡ Luana Curbani,† Timothy A. Parker,§ Jennifer Garcia,§ Linda D. Schultz,§
and Endler Marcel Borges*,†,∥
†
Departamento de Química, Universidade Regional de Blumenau, FURB, Campus 1, Rua Antônio da Veiga, 140, Victor Konder,
89012-900 Blumenau-SC, Brazil
‡
Institute of Bioanalytical Chemistry, Saarland University, Campus B2.2, 66123 Saarbrücken, Germany
§
Department of Chemistry, Geosciences, and Physics, Tarleton State University, Box T-0540, Stephenville, Texas 76402, United
States
∥
Núcleo Biotecnológico, Universidade do Oeste de Santa Catarina, UNOESC, Rua Paese, 198, Bairro Universitário-Bloco K,
89560-000 Videira-SC, Brazil
*
S Supporting Information

ABSTRACT: This experiment describes a simple protocol for

teaching acid−base titrations using potentiometry, conductivity,
and/or photometry to determine end points without an added
indicator. The chosen example examines the titratable acidity of
a red wine with NaOH. Wines contain anthocyanins, the colors
of which change with pH. Importantly, at the equivalence
point, anthocyanins maintain their color, and this effect can be
captured optically using a desktop scanner. RGB-based color
values are obtained from the digitized images using a dedicated
application and employed to generate plots of |v| versus titrant
volume. The end point is the point at which the slope (d|v|/Δ/
dV) of the titration curve is at a maximum; (d|v| is a vector
expression of the color change). The photometric titrations
were carried out using a plastic 96-well immunology plate and a flat-bed scanner, and RGB values were extracted simultaneously
from all 96 wells in less than 5 min from the images using the ImageJ plugin “ReadPlate”. The wine was also titrated using
conventional potentiometric and conductometric techniques, and methods were compared using F-test, t-test, and one-way
ANOVA. The potentiometric titration yields pH versus titrant volume curves, and the equivalence point is where the slope, or
the slope of the derivative curve (dpH/dV), is greatest. The conductometric titration yields conductivity versus titrant volume
curves, and the end point is determined as the volume where the slope exhibits a marked change. All titration and derivative
curves were plotted and analyzed using spreadsheet software.
KEYWORDS: First-Year Undergraduate/General, Second-Year Undergraduate, Graduate Education/Research, Analytical Chemistry,
Laboratory Instruction, Hands-On Learning/Manipulatives, Problem Solving/Decision Making, Titration/Volumetric Analysis,
Spectroscopy, Potentiometry

■ INTRODUCTION
Winemaking is one of the ancient crafts from which the early
science of Chemistry evolved, and it remains an important
industry in modern times. It is also a topic of interest to many
students and has served as the topic for college level courses
reported in this Journal.1−3 A series of experiments in
enological chemical analysis courses has been developed to
develop a better understanding of the relationships between
acid−base, precipitation, oxidation, and complexation equilibria
in wine.4 Enology degrees are offered at many prestigious
universities today.
Wine acidity is a very important property. If a wine is too low
in acidity, it tastes flat and dull, and it is more susceptible to
infection and spoilage by microorganisms.5 If a wine is too high
© XXXX American Chemical Society and
Division of Chemical Education, Inc.
in acidity, it tastes too tart and sour. Usually, the acidity of wine
can vary over wide ranges, depending on grape variety, ripeness
degree, climatic conditions during ripening, soil type, vineyard
position, technique of grape processing, and wine storage.5
Wines contain a complex mixture of organic acids, which
includes tartaric, malic, succinic, acetic, citric, and lactic acids, of
which tartaric and malic acid account for over 90%.6,7 Tartaric
acid is the strongest of these, and significantly influences the pH
and sensory characteristics of the wine.8,9 Therefore, wine
acidity is most often expressed as grams of tartaric acid per 100
Received: November 18, 2016
Revised: June 1, 2017
A DOI: 10.1021/acs.jchemed.6b00891
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Journal of Chemical Education Laboratory Experiment

mL or per L wine,9,10 although measurements based on proton meters are adequate for student laboratories, and commercial
equivalence can also be used.11 scanners and sophisticated software for plotting titration curves
Titratable acidity of wine is generally determined by titration are universally available for determining the end points of
with a strong base. For either red or white wines, the end point titrations.

■
can be determined electronically with a pH meter, and in white
wines the end point can also be detected visually with an EXPERIMENTAL OVERVIEW
indicator such as phenolphthalein.8,10 The acids present in wine
are relatively weak; therefore, titration with a strong base results The goal of this project was to develop a simple photometric
in an end point in the alkaline region of the pH scale. Since titration method based on images obtained with a regular
tartaric acid is the principal acid in wine, quality control targets consumer-grade computer scanner. These results would then
for the end point are between pH 8.2 and 8.4. For example, in be compared to measurements from conventional potentio-
Brazil, the Instituto Adolfo Lutz recommends pH 8.2−8.4,12 metric titration and conductometric titration techniques using
while the American Society of Enologists suggests pH 8.2.13 F-test and t-test methods, or when using all three methods one-
The wine may be titrated either directly or as a solution diluted way ANOVA may be used to compare results.
with deionized water.9 This lab provided students with the opportunity to work with
A less commonly used method for analyzing a mixture of modern imaging techniques for data collection and then use
acids is conductometric titration. This technique monitors the Microsoft Excel to arrange the data in graphs to determine the
conductivity (reciprocal of resistance) of a solution as a titration end point using derivative methods. This, in turn, provided the
proceeds. Conductance varies with the number, size, and charge students the opportunity to compare their data to that obtained
of the ions involved, and also with solvent characteristics. using more traditional analytical methods. Students would find
Therefore, when an ion is replaced by another ion of this lab to be of increased interest because they could use the
significantly different conductance during a titration, the computer to transform digital images into RGB values and
conductivity will change in a linear manner until the apply statistical methods to compare results.
replacement is complete, after which time a plot of conductance Thus, the authors proposed a photometric titration of red
vs titrant volume will change to a different slope due to the wine carried out using a 96-well microtiter plate and digital
continued addition of additional ions of different conduc- imaging using a consumer-grade scanner for application in an
tance.14,15 Darias-Martin et al. analyzed a group of 115 wines undergraduate chemistry laboratory class experiment. At the
start of the titration, the wine test solution exhibited an acidic
using this procedure and found significant correlation with
pH because of the wine’s organic acids. For example, the
other methods.9
potentiometric titration curve in Figure 1a indicated that the
The main chromophores in wine are anthocyanins, and their
initial pH of the red wine solution was 3.1. Addition of titrant
color in solution is directly proportional to their degree of
(NaOH) to the solution slowly increased pH until the solution
ionization, which, for deprotonation reactions, increases with
pH.8 At the end point (pH > 8.0), the chromophores
experience their greatest degree of ionization, and the wine
solution undergoes its most pronounced color change. This can
serve as the basis for a photometric titration. However, in
recent years digital images have been widely applied in
chemistry research16 and education17−31 to replace the
conventional spectrophotometer.
The use of digital images captured using charge-coupled
devices (CCDs) as a novel instrumental detection technique for
titration was first proposed by da Nobrega Gaiao et al.32 The
application required a discontinuity in the titration curve at the
end point which was associated with the same chemical
equivalence as the traditional titration techniques such as
spectrophotometry, potentiometry, or conductometry. Tôrres
et al.8 used digital images obtained from a WebCam to monitor
the titration of red wine; however, the titrations were carried
out using an intricate apparatus that would be difficult to
reproduce in a regular laboratory class. Soldat et al.33
demonstrated that a digital image analysis of a scanned
microplate image could substitute for a spectrophotometer in
several common quantitative procedures, and Christodouleas et
al.34 showed that CCD equipment could be used to perform
sensitive absorbance measurements for liquid samples in a
parallelized manner with accuracy and precision comparable to
microplate spectrophotometers.
All three of the above procedures, potentiometric titration,
conductometric titration, and photometric titration, share
common features that enable each to be performed in a
student laboratory using current technology at a reasonable Figure 1. (a) Experimental data points for titration of 25 mL of wine
cost in an appropriate time frame. Highly sensitive, inexpensive dissolved in 100 mL of water (titrant, 0.098 mol L−1 NaOH). (b) First
(costing less than US$100), hand-held pH and conductivity derivative d(pH)/d(V) of the titration curve.

B DOI: 10.1021/acs.jchemed.6b00891
J. Chem. Educ. XXXX, XXX, XXX−XXX
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lost its buffering capacity, at which point further addition of For example, in Figure 4 each well was digitized, and the pixel
NaOH solution yielded the maximum increase in pH (Figure area for each well was cut out. Then, RGB values were
1a). Thus, the end point was easily determined from the
maximum value of the first derivative of the titration curve
(dpH/dV), as shown in Figure 1b. Spreadsheet data for Figure
1 are shown in Figure 2.

Figure 2. Spreadsheet containing data used to plot Figure 1a and

calculations used to obtain the first derivative plot of Figure 1b.

The photometric experiment recorded color intensity as a

function of titrant addition. In the lab exercise, students
successively added NaOH titrant to a diluted wine sample, and
the pH values were recorded. The resulting data was presented
as a plot of pH versus titrant volume (Figure 1a). For each
volume of titrant added, a small amount of wine solution was
transferred to an individual well of a 96-well microtiter plate as
shown in Figure 3.
This microtiter plate was used to determine the color Figure 4. Color variation (|v|) of red wine after addition of a 0.098 mol
information for the sample for each titrant volume, which was L−1 of NaOH titrant. (a) |v| versus volume of titrant (NaOH, mL). (b)
then illustrated as a plot of color intensity versus titrant volume. First derivative d|v|/dV of the titration curve.

Figure 3. Color variation of red wine after addition of a 0.098 mol L−1 solution of NaOH titrant. Note that all samples are analyzed in triplicate.
Number represents volumes of NaOH added to the wine solution during titration.

C DOI: 10.1021/acs.jchemed.6b00891
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Journal of Chemical Education
obtained. Students then processed these digitized spots to
obtain the exact graphs shown in Figure 4.
As per university policy for all experimental procedures
involving students, all authors in the United States group
underwent IRB training, and an IRB approval form was
submitted to the University Institutional Review Board for
review. The application was approved as Exempt (under 45
CFR 46.101b1), but IRB protocols were followed throughout
the duration of the experiment.
■ EXPERIMENTAL SECTION
Red wine (25.00 mL) was transferred to a 400 mL beaker, and
100.00 mL of distilled water was added. The pH electrode was
placed within the beaker. The solution was then titrated with a
solution of NaOH under magnetic stirring. The United States
group used a 1 L beaker to allow space for an additional
conductivity meter, and used a glass stirring rod. The wine was
titrated with 0.10 mol L−1 solutions of NaOH, which were
previously standardized against potassium hydrogen phthalate.
Grape juice can be substituted if the use of a nonalcoholic
beverage is required. White wine may be used for the
potentiometric and conductometric titrations with no prob-
lems; however, scanning of the white wine plates gave
ambiguous results.
For the 0.1 mol L−1 solution of NaOH titrant, at the
beginning of the titration, the first four additions of titrant were
5.00 mL aliquots for the protocol used in Brazil. Then, 1.00 mL
aliquots of titrant were repetitively added, as small additions of
titrant near the equivalence point to help to precisely determine
the end point. The United States group uniformly added 2.00
mL aliquots throughout the titration.
For each NaOH addition, the measured pH (or con-
ductivity) value was recorded as a function of the added titrant.
Then, three small samples of the wine solution were placed in a
96-well microtiter plate, and the plate was scanned to generate
a 600 dpi color image in .jpg format (Figure 3). In laboratory
classes carried out in Brazil, aliquots were delivered into wells
using a Pasteur pipet, and students were instructed to deliver
equal volumes to each well. The aliquots delivered using
Pasteur pipettes at each well were slightly imprecise; however,
this did not affect results.
RGB values were extracted from digital images using ImageJ’s
plugin ReadPlate, which extracts simultaneous RGB values from
all 96 plates (see Supporting Information).
The most common scale for color images is RGB (red green
blue). In this color space, each pixel is defined by the intensities
of red (R, λ≈ 630 nm), green (G, λ ≈ 545 nm), and blue (B, λ
≈ 435 nm).35 For this application, the RGB channels for each
sample well on the digital image were captured and average
values of each channel calculated. Then, a vector norm |v| was
determined using R, G, and B (the normalized average intensity
values obtained from the differences between the RGB channels
of sample wells and the RGB channels from blank, empty,
spots).
The United States group experimented with both Paint using
eyedropper and ImageJ for isolating spots, but ultimately
decided to use ImageJ because of its more representative area.
Laboratory classes carried out in Brazil, initially, used R-project
to extract RGB values from the wells, but ultimately settled on
ImageJ due to its greater convenience in extracting data from
multiple wells simultaneously. Details on its use can be found in
the student instructions in the Supporting Information.
D DOI: 10.1021/acs.jchemed.6b00891
Laboratory Experiment
The laboratory in the United States also simultaneously
monitored solution conductivity as the titration progressed, and
a sample conductometric titration curve is shown in Figure 5.
Figure 5. Sample conductometric titration curve for red wine. The
equivalence is between red squares, in y = 1.8.
The shape of these was markedly different from that observed
in simpler systems, such as sulfates or strong acids,36,37 and
similar to those for mixtures of weak acids,37 such as wines, as
reported by Darius-Martin et al.9 The equivalence point is
represented by the intersection of the first two trendlines,
which represent titration of the tartaric and malic acids. The
dilution correction factor recommended for most applications
could not be effectively applied because of the volume changes
introduced by sample removal for photometric analysis, which
also resulted in irregular trendlines. However, the relatively
large solution volumes used minimized the dilution effects, and
stand-alone conductometric titrations of red wine done using
this procedure did not show significant differences in
equivalence point volumes when the correction factor was
introduced. It should also be noted that the US lab used more
concentrated NaOH solutions for their titrations (0.1314 M
compared with 0.098 M in Brazil). This resulted in smaller
titrant volumes, and sharper end points, and also reduced
dilution effects.
Laboratory practices describing SO32− analyses in white
wines39,40 were reported in this Journal, but it is the first
laboratory practice to deal with potentiometric, photometric,
and conductometric titrations.
■
HAZARDS AND SAFETY
Care must be taken in handling all chemicals. Sodium
hydroxide is corrosive to skin and eyes and an irritant to the
respiratory tract if inhaled. Potassium hydrogen phthalate is a
respiratory irritant and harmful if swallowed. Safety glasses and
rubber gloves should be worn when manipulating the
chemicals, and proper clothing and footwear should be worn
at all times.
■ RESULTS AND DISCUSSION
In the first semester of 2016, this laboratory experiment was
carried out in Brazil with second-year chemistry, chemical
engineering, and production engineer students. It was typically
finished in approximately 3.5 h with 18 students working
individually or in groups of three, as needed. During the first 90
min of class, a group of three students usually performed three
J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education Laboratory Experiment

titrations. These included performing the NaOH stand- average, and n is the number of measures. The tcritical may found
ardization process, recording the pH values as a function of in static tables or using the Microsoft Excel equation TINV
volume of titrant, pipetting a small volume of wine solution into (probability; degree of freedom) = TINV (0.05; 10).
a separate well of the microtiter plate after each titrant addition,
|m1 − m2| n1n2
and scanning the finished plate. After the measurement process, t=
the data treatment and interpretation required approximately 2 spooled n1 + n2 (2)
h. During this period, each member of the group extracted RGB
values using ImageJ’s plugin ReadPlate. The computed RGB s12(n1 − 1) + s22(n2 − 1)
values were then imported into a spreadsheet, and the end spooled =
point was calculated using the first derivative method. n1 − n2 − 2
In May 2016, five groups titrated the same bottles of wine, In the example shown, the potentiometric titration (Figure
using potentiometric and spectrophotometric titrations. Results 1b) gave an end point of 24 mL, while a spectrophotometric
obtained from these groups are summarized in Table 1. titration (Figure 4b) provided a value of 23.5 mL, reflecting
close agreement between both methods (6.73 and 6.88 g of
Table 1. Comparison of Student Group Results for tartaric acid per liter, respectively).
Potentiometric and Photometric Titrimetric Analysis of a In July 2016, the US group carried out this experiment in a
Red Wine college chemistry II laboratory. These students were only
Group Results by Method (g) Tartaric Acid (L−1) enrolled in their second semester of an introductory chemistry
Number Photometric Potentiometric course, but they had already completed the acid/base portion of
1 7.16 7.30 the lecture and performed lab experiments involving potentio-
1a 7.30 b metric titration, use of a spectrophotometer, and use of Excel
2 6.73 6.88 spreadsheets in a graphing application similar to the
2a 6.73 b conductometric titration curve, so this experiment was not
3 7.88 7.88 inappropriate for their level of knowledge. Also, the summer
4 7.88 7.88 laboratories periods were longer than those in the regular
5 7.88 8.16 semester, 4 h compared to 3 h.
Av (N = 7) 7.37 7.62 Students were given the laboratory handout included in the
SD (N = 7) 0.48 0.52 Supporting Information 1 week prior to performing the lab to
a
Groups 1 and 2 did potentiometric titrations two times, thus N = 7. familiarize themselves with the procedures, and this was also
b
Not measured. Thus, N = 5 for photometric titration. reviewed by the instructor at the beginning of the lab, which
was set up as a “single pot” experiment with pH and
The spreadsheet obtained by each group of students is in conductivity meters in the same beaker. As each 2.00 mL
Google Drive.38 To compare the standard deviations, an F-test increment was added, the pH and conductivity were recorded,
can be conducted to see whether the standard deviation from and three samples of 0.25 mL were removed and placed in
the potentiometric analysis is significantly different from the numbered wells of the microtiter plate. Thirty-nine students
standard deviation from the spectrophotometric analysis. F was worked in teams of five or six, with one pair on each team
calculated using eq 1; the larger variance was put in the responsible for collecting a different data set. Data collection
numerator (s1) to obtain F > 1, F = (0.52)2/(0.48)2 = 1.16. required about 3 h. Then, the groups relocated to the computer
lab to calculate their results, which took about an hour and
s12 were later presented in a written group lab report. Their results
F=
s22 (1) are summarized in Table 2.
One-way ANOVA (analysis of variance) compares the means
Within the confidence level of 95%, we found Fcalculated = of two or more independent groups in order to determine
1.507 < Fcritical = 4.53 (4 degrees of freedom and 6 degrees of
freedom) and concluded that the differences between the Table 2. Comparison of Student Group Results for
standard deviations were not significant. The Fcritical may be Potentiometric, Conductometric, and Photometric
calculated using the Microsoft Excel equation FDIST Titrimetric Analysis of a Red Wine
(confidence interval; degrees of freedom s1; degrees of freedom
s2) = DISTF (0.05; 4; 6). Results by Method of Analysis, g Tartaric Acid L−1
The F-test may be carried out using the Analysis ToolPak in Group Number Potentiometric Conductometric Photometric Average
Excel to find P′(F ≤ f) one tail = 0.41, where it also may be 1 5.84 5.52 5.13 5.50
calculated using Microsoft Excel function FDIST (F; degrees of 2 5.57 5.13 5.92 5.54
freedom s1; degrees of freedom s2) = FDIST (1.507; 4; 6). 3 5.72 a 5.92 6.25
The t-test compares the means of two independent groups to 4 5.33 5.92 5.13 5.46
determine whether there is statistical evidence that the 5 5.09 6.71 5.13 5.64
associated population means are significantly different. The t- 6 5.33 5.13 5.13 5.19
test assuming equivalent standard deviations was carried out 7 5.33 6.31 5.13 5.59
using Analysis ToolPak in Excel, within the confidence level of Av (N = 7) 5.46 5.79 5.35 5.53
95%, to find tcalculated = 0.83 < tcritical = 2.22 (10 degrees of SD (N = 7) 0.26 0.64 0.38 0.43
freedom), P(T ≤ t) two tail = 0.45, and concluded that tartaric
acid concentrations found with potentiometric and spectro- a
Group 3 experienced excessive problems due to a weak battery in
photometric titrations were equivalent. The tcalculated may also be their conductivity meter; that result was excluded from this table, thus
calculated using eq 2, where s is the standard deviation, m is the N = 6.

E DOI: 10.1021/acs.jchemed.6b00891
J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
whether there is statistical evidence that the associated
population means are significantly different. Using the Analysis
ToolPak in Excel, within the confidence level of 95%, we found
Fcalculated = 1.61 < Fcritical = 3.59 (2 degrees of freedom; 17
degrees of freedom) showing that results obtained with the
three methods were equivalent at a 95% confidence interval.
Agreement among the three titrimetric methods was
generally good, although the conductometric titration results
tended to be slightly higher than the other two. This was also
noted by Darias-Martin et al.9 who surmised that this method
actually gave the total acidity, rather than the titratable acidity. It
should be noted that the average value of the titratable acidity
obtained by the students of 5.53 g tartaric acid/L is in good
agreement with that obtained from the analysis of 65 red wines
from the Canary Islands of 5.49 g tartaric acid/L.9
■
CONCLUSIONS
This paper presented a simple photometric acid−base titration
method for a student lab in the chemistry undergraduate
curriculum, implementing digital imaging using a consumer-
grade computer scanner, for the determination of titratable
acidity of red wines without the use of dye indicators. F-test, t-
test, and one-way ANOVA methods were used to compare the
data from the new spectrophotometric titration method with
those of conventional potentiometric and conductometric
titrations, which also enabled the students to gain experience
in those techniques. Anthocyanins present in wines exhibit
different colors at different pH values as a result of different
ionization equilibria of these molecules with changing pH. This
color change was utilized for detection. At the end point of the
titration, anthocyanins reached their maximum change of color,
after which the color remained almost constant with further
increase of pH. The end points of the three titration methods
were determined from the slopes of the color (photometry),
pH (potentiometry), and conductivity (conductometry)
changes with added titrant volume, where the first derivative
of the titration curves showed a curve maximum. In addition to
the titration experiments, the students learned the basics of
image processing, mathematical first derivative methods, and
statistical comparisons between methods.
During the implementation of the new titration experiment,
we observed that the students became interested in the modern
image-processing spin added to the experiment. Because
imaging processing techniques have recently received signifi-
cant attention in many areas popular with young people, we
believe this new variation of a classic experiment will greatly
increase the learning experience and interest of the students.
Even statistical methods can be more easily communicated
when they are implemented as part of a spreadsheet-based
exercise.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available on the ACS
Publications website at DOI: 10.1021/acs.jchemed.6b00891.
Student laboratory handout for the combined (potentio-
metric, conductometric, and photometric) titration
experiment, notes for instructors, and details regarding
materials and hazards (PDF, DOCX)
Microsoft Excel template that can be used to import
results from ImageJ to Microsoft Excel (XLSX)
F DOI: 10.1021/acs.jchemed.6b00891
■
Laboratory Experiment
AUTHOR INFORMATION
Corresponding Author
*E-mail: marcelborgesb@gmail.com.
ORCID
Endler Marcel Borges: 0000-0002-9260-3639
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors acknowledge financial support and fellowships
from the Brazilian agencies FAPESC (Fundaçaõ de Amparo a
Pesquisa do Estado de Santa Catarina), CNPq (Conselho
́
Nacional de Desenvolvimento Cientifico e Tecnológico, Grant
number 402226/2016-0), and CAPES (Coordenaçaõ de
Aperfeiçoamento de Pessoal de Niveĺ Superior), and the
German Alfried Krupp von Bohlen und Halbach-Stiftung. The
financial assistance of The Robert A. Welch Foundation,
Chemistry Departmental Grant AS-0012, is gratefully acknowl-
edged.
■
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G DOI: 10.1021/acs.jchemed.6b00891
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