Laboratory Experiment

pubs.acs.org/jchemeduc

Introduction to Validation of Analytical Methods: Potentiometric

Determination of CO2
A. Ricardo Hipólito-Nájera,† M. Rosario Moya-Hernández,† Rodolfo Gómez-Balderas,*,†
Alberto Rojas-Hernández,‡ and Mario Romero-Romo§
†
Laboratorio de Fisicoquímica Analítica, UIM, FES−Cuautitlán, UNAM, Campo 4. Km 2.5 Carretera Cuautitlán Teoloyucan, 54740
Estado de México, México
‡
Departamento de Química, Á rea de Química Analítica, Apdo, UAM−Iztapalapa, Apdo. Postal 55−534, 09340 Iztapalapa, Cd.
México, México
§
Departamento de Materiales, Á rea Ingeniería de Materiales, UAM−Azcapotzalco, Av. San Pablo 180, Colonia Reynosa−Tamaulipas,
02200 Cd. México, México
*
S Supporting Information

ABSTRACT: Validation of analytical methods is a fundamental subject

for chemical analysts working in chemical industries. These methods are
also relevant for pharmaceutical enterprises, biotechnology firms,
analytical service laboratories, government departments, and regulatory
agencies. Therefore, for undergraduate students enrolled in majors in the
field of chemistry, learning validation of methods and chemometrics is
essential. The following article presents the development of a laboratory
experiment to validate a method to determine CO2 by means of
potentiometry. This experiment will allow students to approach a subject
that may be part of their professional activities. The validated method was
applied to determine CO2 in commercial carbonated beverages. A
teaching activity on method validation was proposed and implemented for
senior students. After its application, students were able to improve significantly their knowledge on the topic. The
potentiometric technique, underlying the validated method, has the advantage of being easy to apply and accessible in almost any
undergraduate laboratory. Furthermore, the required reagents are available practically in any chemistry department. The shown
procedures might be executed as laboratory activities in analytical chemistry due to their overall simplicity and rapidity.
KEYWORDS: Analytical Chemistry, Upper-Division Undergraduate, Ion Selective Electrode, Potentiometry, Chemometrics,
Laboratory Instruction

■ INTRODUCTION
To fulfill more stringent local and international regulations, and
teaching. This might hinder students in learning the essentials
of validation. Therefore, in this work, we provide students with
to maintain worldwide competitiveness, global firms must a single appropriate validation method to analyze CO2 in
ensure the highest quality control of their products. Validation carbonate drinks. The presented method takes parameters and
of an analytical method is a documented process used to recommendations from different guides. Here, such parameters
confirm that the procedure employed to conduct a specific test and recommendations are unified with a pedagogical intention.
is adequate for the intended application and used to assess the Scholarly papers that include some aspects of validation have
quality, reliability, precision, and consistency of analytical been recently published. For instance, Stitzel et al.5 worked on
determinations.1−3 Skills related to the validation of analytical an internal standard calibration and then a validation of a high−
methods are fundamental for good practices in chemistry. performance liquid chromatography (HPLC) method by
Although there are courses in chemometrics, method develop- comparison to a certified reference material. Usher6 gave an
ment, verification, and validation, they are rather intended to introduction for students to some validation procedures on
professional chemists. Thus, it is highly desirable for students of liquid−liquid extraction and HPLC methods comprising
chemistry to develop these skills while enrolled in the calibration curves and recovery evaluations. This work is an
university. example of a partial validation with a small set of parameters. By
There are numerous approaches to validate analytical working with cold vapor atomic absorption spectroscopy,
methods; all of them are acceptable as long as they are
scientifically supported.2 Guides and technical publications on Received: April 5, 2017
method validation are abundant;4 however, the aim of those Revised: June 16, 2017
texts is different from direct application on undergraduate
© XXXX American Chemical Society and
Division of Chemical Education, Inc. A DOI: 10.1021/acs.jchemed.7b00252
J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education Laboratory Experiment

Kristian7 determined the linearity and limit of detection for

analyzing referenced materials. Recommendations on recovery
studies are given to students. Earlier, Donais8 had carried out
studies of the limit of detection and precision for flame atomic
absorption spectroscopy, and students studied the significance
of precision in data evaluation. Homem9 reported how students
validated a method to analyze amoxicillin in river waters by
means of liquid chromatography−mass spectrometry. Valida-
tion of this particular analytical method included drawing a
calibration curve, determining detection and quantification
limits, as well as precision and accuracy of the method.
Literature shows important aspects of method validation, but
their implementation depends on advanced instrumental
analytic techniques, which might be a shortcoming for average
undergraduate laboratories. In contrast, the laboratory experi-
ment presented here relies on the broadly accessible
potentiometric technique.
To accomplish the validation of a particular analytical Figure 1. Parts and schematic view of a gas sensing electrode
method, several experimental procedures are usually required.3 responsive to CO2. This probe fundamentally works as a pH electrode.
For example, sample preparation, plotting calibration curves,
and data analysis are core procedures of method validation.
Depending on the analytic application of the method, a set of of a laboratory experiment in team-working guided sessions.
parameters is chosen to validate its performance1 such as This experiment has been carried out with students enrolled in
precision, limit of quantification, limit of detection, etc. undergraduate programs, at the Facultad de Estudios
Teaching validation of analytical methods could be addressed Superiores Cuautitlán of the Universidad Nacional Autónoma
by performing a narrow validation and calculating only one de México, on chemistry but can be extended to related majors
parameter such as accuracy (and its uncertainty) across several such as industrial chemistry, chemical engineering, pharmacy,
food engineering, etc.

■
techniques, for example, spectroscopy, voltammetry, or
potentiometry. Also, a partial validation is feasible by
determining a selected set of parameters for only one MATERIALS, EQUIPMENT, AND REAGENTS
technique. Certainly, this second approach requires less Carbon dioxide sensing probe, supplied with membrane kit,
equipment and laboratory resources. Because full validations sodium hydrogen carbonate internal electrolyte solution
are beyond the teaching goals in a scholarly environment, (Mettler Toledo), combined pH/conductivity meter (Seven-
partial validations are more appropriate due the amount of Multi S47, Mettler Toledo), recirculating bath (FC 6, SEV),
resources involved in their execution. Therefore, we have deionizer (Pure Lab Classic, ρ = 18.2 MΩ cm). Sodium
chosen a partial potentiometric validation, by means of a gas hydrogen carbonate (analytical grade, JT Baker), dihydrate
sensing electrode, taking into account that this is one of the sodium citrate, (ACS grade, JT Baker), concentrated hydro-
most easy and accessible techniques in almost any laboratory. ́
chloric acid, (Quimica Meyer), sodium chloride (analytical
The beginning of the selective electrodes started with the ́
grade, Reactivos y Productos Quimicos Finos, México), sodium
development by Walter Nernst of the hydrogen H+(aq)/H2(g) ́
hydroxide (analytical grade, Quimica Meyer), anhydrous
dibasic sodium phosphate (ACS grade, Fermont).

■
electrode. One of the most important selective electrodes is the
glass membrane electrode, also known as the pH electrode.10
Later, in 1953, Stow11,12 developed a gas sensing electrode for HAZARDS
CO2. As can be seen in Figure 1, the CO2 sensing electrode has Concentrated HCl and NaOH are corrosive, toxic, and
a gas permeable membrane and a standard NaHCO3 internal irritants; work under a fume hood. Sodium citrate and sodium
aqueous solution. The CO2 gas to be quantified diffuses to the phosphate are harmful if swallowed and eye irritants. Wear
laboratory coat, safety glasses, and gloves.

■
internal solution across the membrane generating the ionic
species H+, HCO3−, and CO32−. As concentration of these
species change in the internal solution, pH variations are VALIDATION OF A POTENTIOMETRIC METHOD
detected by an inner pH electrode 11−13 (Figure 1). FOR DETERMINATION OF CO2
Applications of this sensing electrode include CO2 measure- As mentioned before, validation relies on evaluating a set of
ments in blood,14 CO2 generated from metabolic processes in suitable chosen parameters that gives the performance of a
plants,15 and determination of urea16 and oxalate.17 Moreover, particular method.1 Thus, to accomplish the validation of the
quantification of CO2 is a fundamental element of quality potentiometric determination of CO2 in a solution, we have
assurance in fields such as agricultural industry and food.18 For chosen the following parameters, regarded as the most
instance, CO2 detection and quantifications are carried out by important for validating analytical procedures:1 linearity
practically any soft drinks, mineral water, and breweries range, limit of quantification (LOQ) and limit of detection
producers.17,19 (LOD), precision, robustness, sensitivity, accuracy, and
The aims of the present work are first, to present the specificity. Parameter definitions, as well as experimental
performance of a series of parameters needed to validate a protocols for their determination, are provided in detail in
potentiometric method to quantify CO2 in carbonated drinks. the Supporting Information. It is important to mention that a
Second, demonstrate the application of the validated method. diligent experimental design must be applied in the whole
Finally, propose, implement, and corroborate the achievements validation procedure.
B DOI: 10.1021/acs.jchemed.7b00252
J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education Laboratory Experiment

Linearity Range, Limit of Quantification, and Limit of It is interesting to analyze the origin of nonlinearity at the
Detection extremes of the calibration curve. The lowest section in Figure
Linearity defines the ability of the method to obtain test results 2 is horizontal because of the low concentrations of CO2. In
proportional to the concentration of analyte. In case of a CO2 this region, a small amount of CO2 diffuses across the
sensing electrode, the LOQ is defined as the beginning of the membrane, having no effect on the pH of the internal solution.
linear range where the Nernstian response starts. For some Then the upper section of the curve is again horizontal due to
authors,17 this is the lowest point for making useful the limited range of response of the CO2 saturated internal
determinations (Figure 2). The standards should be prepared solution and the glass electrode.17
Once the linearity range has been determined, the LOQ17
and LOD13 can be obtained being 8.98 ± 1.1 mg L−1 and 1.48
± 0.04 mg L−1, respectively. See details in Supporting
Information, sections 1.3 and 1.4. Performance parameters of
the calibration curve are shown in Figure 2 and compiled in
Table 1.

Table 1. Results of Parameters of the Method

Parameter Acceptability Criteria Experimental Results
Linearity R2 ≥ 0.98a,b R2 = 0.9994
Slope = 54.02 ± 0.34 mV
Range: 8.89−280.92 mg L−1
LOQ 8.98 ± 1.1 mg L−1
LOD 1.48 ± 0.04 mg L−1
Precision r&R < 10%c r = 5.94 mV
R = 2.38 mV
r&R = 3.00%
Robustness |di| ≤ 3%d Temperature: |di| = 2.8%
Buffer solution: |di| = 3.4%
Figure 2. Variation of the potential E as a function of log [CO2]TOTAL
Open beaker: |di| = 6.5%
for the three sets of determinations. Linearity range, LOQ, and LOD
are indicated. [CO2]TOTAL is given in mg L−1. Sensitivity Slope ≈ 59.16 mVe 54.02 ± 0.34 mV
Accuracy CV ≤ 5%d CV = 2.73% and 2.00%
PRec = 99.7% and 99.6%
carefully to observe the point where the calibration line Specificity 100% must be included in PRec = 96.7 ± 6.2%
becomes curved. Thus, the LOQ becomes independent of the PRec
chosen concentrations to prepare the standards. a
See ref 1. bSee ref 17. cSee ref 25. dSee ref 21. eSee ref 13.
On the basis of the IUPAC recommendation,13 the practical
lower LOD may be taken as the concentration of CO2 at the
Precision
intersection point of the extrapolated linear midrange and final
low concentration level segments of the calibration curve Precision is described by two components: the smallest
(Figure 2). To establish our interval of linear analytical expected precision and the largest encountered measured
response and to set up the LOQ and LOD, an experimental precision, known as repeatability (r) and reproducibility (R),
calibration curve was plotted (three times) by measuring the respectively.22,23 These components were evaluated via a
potential (E) of standards of CO2 with concentrations ranging standard study of two-way analysis of variance (ANOVA)
from 8.88 × 10−5 to 2.81 × 104 mg L−1. NaHCO3 was used as without interactions. It has been recommended to prepare ten
primary standard to prepare standard solutions, and to adjust different concentrations (c = 10), in duplicate (m = 2), and
the pH value to 4.5 a 1.73 M citrate/citric acid buffer solution perform readings by three different analysts (a = 3).24−26 For
was used (see Supporting Information, section 1.1). this purpose, concentration ranged from 5.02−891.69 mg L−1.
Visually we can establish that the calibration curve, presented Notice that concentrations values outside the linearity range
in Figure 2, tends to be linear in a defined interval of have been used to challenge the validation beyond its linearity
concentrations. To define the linearity range, a search within limits:
different data sets must be conducted by choosing the set that r = 5.15 MSE (2)
gives the best R2 and closely follows the Nernst law. Linearity
range is established from Figure 217,20 by applying least-squares MSA − MSE
fitting to the experimental potentials; thus, R = 5.15
cm (3)
E = −(210.55 ± 0.61) + (54.02 ± 0.34) log[CO2 ]TOTAL In eqs 2 and 3, MSE and MSA stand for mean square error
(1) and mean square of the analyst, respectively.24−27 For normally
where E is obtained in mV. distributed data, the value 5.15 defines 99% of the space under
Linearity is observed from 8.89−280.92 mg L−1 (Figure 2), the normal curve.28 The raw data, the explanation for the use of
and the obtained potentials display a high determination ANOVA without interaction and the evaluation of r and R
coefficient of R2 ≥ 0.99, denoting also a Nernstian behavior.21 through ANOVA analysis are found in Table 5−8 of
Furthermore, at 97.5% significance level the plot exhibits a Supporting Information, section 1.5.
slope value with a confidence interval (CI) of 54.02 ± 0.34 mV The r and R are calculated by means of eqs 2 and 3.24,26
(eq 1). Refer to Supporting Information, section 1.2. Results for r and R are shown in Table 1; please notice that
C DOI: 10.1021/acs.jchemed.7b00252
J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education Laboratory Experiment

these parameters have the same units of measurement as the (or activity) of the active species (aA) with charge zA, which is
original observation. Thus, r = 5.94 mV > R = 2.38 mV, and this expected to have a slope of 59.16/zA mV per unit change of
result might indicate that the equipment needs adjustments or log(aA) at 298.15 K. Both recommendations point out that the
that the measurement processes should be improved.25 slope of the potential versus logarithm of the concentration
Finally, to evaluate the inherent precision of the method curve gives the method sensitivity. Thus, this parameter is
r&R, eq 4 is applied:29,30 already established from the linearity range fitting equation (eq
1), its value is 54.02 ± 0.34 mV. See Supporting Information,
r &R = r 2 + R2 (4) section 1.7.
The value of r&R = 6.40 mV, to obtain the percentage is Accuracy
necessary to make a relation with the total variation obtained Accuracy is the quantity resulting from the differences between
with ANOVA as 100%; the percentage of r&R is 3.00%. the average of a series of measurements, or an individual
Because the percentage of r&R < 10%, the measurement measure, and the accepted value as the true or correct value for
system is acceptable.24−26 a measurement.1,21,23 The accuracy is determined by means of
Robustness the percentage of recovery, with an experimental calibration
The robustness of an analytical procedure is a measure of its curve obtained in the linear interval of E versus log[CO2]TOTAL.
capacity to remain unaffected by small, but deliberate variations To do so, the potential of two solutions of two known
in method parameters. Robustness provides an indication of the concentration, 88.61 and 132.91 ppm, was measured in
method reliability during normal usage.1 For determining sextuplicate.1,21,22 Results of experimental determinations, the
robustness, one deliberate modification at the time in the average of PRec, and the coefficients of variation, are compiled
experimental conditions must be carried out.1 We modified in Table 1, see Supporting Information, section 1.8.
temperature, nature of the buffer, and beaker either closed The average recovery for two different concentration levels
(with lid) or open (without lid). For the regular conditions of 88.61 mg L−1 and 132.91 mg L−1, are 99.7% and 99.6% with
the experiment, T = 25 °C, citrates buffer in a closed system, a coefficients of variation of 2.7% and 2.0%, respectively.
calibration curve in the linearity range was built reading three Consequently, the accuracy of the method is acceptable.1
standards. Then this curve is employed to estimate, again in Specificity
triplicated, a standard analyte of a known concentration of
Specificity is the capacity of the analytical method to determine
[CO2]TOTAL = 50 mg L−1, under regular and altered conditions:
exactly and specifically the analyte of interest, in the presence of
T = 38 °C or buffer of phosphates (pH = 4.5) or open system.
After the average percentage of recovery (PRec) was other components, in a sample matrix under the established test
obtained, by means of eq 5, for the regular and altered conditions.1 To know the specificity of the method for the
conditions, PReco and PReci, respectively, the absolute objective it is intended for, it is necessary to execute
difference |di| = |PReci − PReco| was determined to evaluated determinations in the presence of the matrix. In this particular
the robustness of the method: the smaller the |di|, the more case, carbonated drinks were degassed to eliminate the CO2 as
robust the method. It has been established18 that, after altering much as possible by heating and ultrasound−bath agitation;
a factor, a |di| ≥ 3% indicates the robustness of the method is this matrix was then employed to prepare NaHCO3 standard
compromised. Table 1 compiles the results obtained for the solutions. The specificity parameter determined in such a way
described challenges to the method; see Supporting Informa- shows the viability of using a calibration curve in water to
tion, section 1.6. quantify the CO2 in carbonated drink samples. Table 1 displays
the experimental results for evaluating the specificity of the
[CO2 ]TOTAL exp method, Supporting Information, section 1.9.
PRec = × 100 As it can be observed in Table 1, the average value of the
[CO2 ]TOTAL Standard (5)
recovery is 96.7% with a coefficient of variation of 6.2%. Such a
Thus, the factor that has more impact on the recovery is low value for the recovery might indicate that the treatment of
opening the beaker (6.5%), followed by switching the buffer the matrix introduces unexpected variability on the results.

■
solution from citrates to phosphates (3.4%) and the change in
temperature (2.8%). Therefore, because the |di| of the APPLICATION OF THE VALIDATED METHOD:
temperature and the buffer solution did not go over 3%, DETERMINATION OF CO2 IN CARBONATED
these factors can be modified under regular working conditions DRINKS
and would have a minimum effect on the overall performance
of the method. In contrast, CO2 determination is revealed to CO2 Calibration Curve
depend strongly on the open/closed condition of the system.
As it has been discussed, a central element for analyte
Consequently, it is not recommended to modify this parameter
determinations is the calibration curve. Unknown samples are
for routine application of the method.21
read in the calibration curve to find the corresponding
Sensitivity concentrations. Calibration curve validity is based on an
Following the EURACHEM Guide,1 the analytical sensitivity is accurate and precise recovery for known samples, which are
the change in instrument responses, which corresponds to a independent of the standards, prepared in the same manner as
change in the measured quantity (for example an analyte the unknown.
concentration), that is, the gradient of the response curve. In this particular case, we prepared a series of solutions by
When the potentiometric method sensitivity needs to be dissolving NaHCO3 in deionized water. The covered range of
estimated, IUPAC13 recommends to observe the Nernstian concentrations went from 24.98 to 149.91 mg L−1, which lies in
response in the plot of the potential difference of the ISE (ion- the linearity interval. The E was then measured for each
selective electrodes) against the logarithm of the concentration solution, three times, and the average plot was used for the
D DOI: 10.1021/acs.jchemed.7b00252
J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education Laboratory Experiment

determinations. More details of the calibration curve are found acquired knowledge. At the end of the last session, a final quiz
in Supporting Information section 2. was applied to evaluate the global learning. Results of the
Sample Preparation and Determinations grades on these quizzes showed that the scored average
substantially increased from 2.3, 3.8 to 6.5 on a scale of zero to
Several different samples were prepared from the four ten.
commercial cola carbonated drinks retailed in Mexico City, Finally, Table 2 compiles all the results obtained by the
labeled as X, X Light, Y, and Y Light. The drinks used in this students for determining CO2 content in carbonated drinks;
experimentation were from 600 mL of polyethyene terephtalate
(PET) bottles. As soon as the drinks are opened, due to the Table 2. Students Experimental Results and CO2 Content in
change of pressure, they start to effervesce, and CO2 escapes. Carbonated Drinks
Thus, the pH must be promptly adjusted to a value greater than
10 for the CO2 to be transformed in CO32− and stay in the Working Group [CO2] in the Bottle (g L−1)a
solution during manipulations. 1 6.36 ± 0.11
Determination of CO2 concentration was successfully 2 6.17 ± 0.08
achieved using the validated method; it produces acceptable 3 6.31 ± 0.08
results because the values are in good agreement to those 4 6.72 ± 0.25
reported for CO2 in carbonated drinks (4−8 g L−1).31,32 Table 5 6.01 ± 0.19
15 in Supporting Information section 2 compiles these results. 6 6.80 ± 0.36
An evident advantage of this potentiometric method is its a
Average on triplicated measurements.
inherent rapidity for the determination of a large series of
samples. Once the method has been validated, it is possible to
carry out several measurements of the problem samples, with notice that treatment of the samples implies dilution to adjust
only one calibration curve, in minutes for routine use. the concentration to the linearity range of the method. Their

■ LEARNING EXPERIENCE
results are consistent with our previous results showing that
students were able to apply a validated method to quantify the
analyte in commercial samples.

■
Bearing in mind that method validation is a subject scarcely
treated in regular courses of chemistry, a didactical approach
CONCLUSIONS
was designed for teaching validation to students on this field by
employing the same set of validation parameters already An analytical method aimed to quantify the amount of CO2 in
discussed. We employed the CO2 gas sensing potentiometric carbonated drinks has been successfully proposed and validated,
determination previously discussed for introducing the topic on following the recommendations of international regulatory
method validation as well as the application of the validated agencies. As the validation procedure relies totally on
method for CO2 quantification in carbonated drinks. potentiometry, it is easily affordable to almost any chemistry-
Initially, to develop the laboratory experiment, we invited 19 teaching laboratory, which allows the teachers to include
students enrolled in the B.Sc. on either chemistry or industrial method validations as an important subject to be taught. More
chemistry, in the Facultad de Estudios Superiores Cuautitlán sophisticated and costly techniques could be validated by
(Universidad Nacional Autónoma de México); this group was evaluating basically the same set of validation parameters
subdivided in six smaller working groups. The experiment was presented here. A learning experience of method validation was
divided in three laboratory sessions. proposed and implemented in a team-working guided teaching
Working with potentiometry is rather simple; therefore, skills activity to introduce the topic in undergraduate level.
of the students were not evaluated prior to experimentation, Evaluation of the learning experience shows that students
but a series of good laboratory practices were recapped. Each improved their knowledge on method validation. The present
laboratory session proceeded with a short lecturing on the work showed that once students have validated the method,
principles of method validation and the parameters to be they are capable to satisfactorily quantify CO2 in real
commercial samples.

■
evaluated during the session, followed by hands-on guided
sessions for data collection. After the potentiometric readings
were performed, the students input the collected data in Excel ASSOCIATED CONTENT
worksheets previously prepared for statistical data processing *
S Supporting Information

(raw data as well as processed data are included in Supporting The Supporting Information is available on the ACS
Information, section 3). The instructor assisted students on the Publications website at DOI: 10.1021/acs.jchemed.7b00252.
analysis and interpretation of results for each evaluated Comprehensive information on solution preparation and
parameter by focusing on how the results could impact the data treatment, usable by teacher assistants or professors,
validation of the whole method. Lecturing (approximate 15 to conduct the experiment (PDF, DOCX)

■
min) and total data collection took about 12 h, divided on three
laboratory sessions (from 3 to 4 h), preparation of standard
AUTHOR INFORMATION
solutions is not considered in this time.
To explore the pedagogical effectiveness of the laboratory Corresponding Author
experiment, we compared the results of a series of the applied *E-mail: rodolfo.gomez@unam.mx.
quizzes. These quizzes are available in Supporting Information, ORCID
section 4. Before the experimental sessions, students took
individual quizzes on validation of analytical methods to assess
Rodolfo Gómez-Balderas: 0000-0002-3293-1841
their previous knowledge on the subject. After lecturing and Notes
experimental session, a final quiz was applied to assess the The authors declare no competing financial interest.
E DOI: 10.1021/acs.jchemed.7b00252
J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education

■
Laboratory Experiment

ACKNOWLEDGMENTS (20) Guidelines for Collaborative Study−Procedures to Validate

Characteristics of Methods of Analysis, Appendix D. In Official
The authors are grateful to PAPIME(UNAM) Grant No. Methods of Analysis of AOAC INTERNATIONAL; AOAC Interna-
203911 and PIAPI (FESC-UNAM) Grant No. 1629 for tional: Rockville, MD, 2015; pp 1−11.
partially financing this work. A.R. H.-N. thanks the PAPIME ́
(21) Colegio Nacional de Quimicos Farmacéuticos Biólogos México,
program scholarship to pursue his major on chemistry. We are A.C. Guiá de Validación de Métodos Analiticos,́ 2nd ed.; Colegio
in debt with all the students that generously worked in the ́
Nacional de Quimicos Farmacéuticos Biólogos México: México City,
project. México, 2002.

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(22) Compendium of Chemical Terminology; Gold, V., Loening, K. L.,
REFERENCES McNaught, A. D., Shemi, P., Eds.; Blackwell Scientific: Melbourne,
1987.
(1) Eurachem Guide: The Fitness for Purpose of Analytical Methods − A (23) ISO 3534−1:2006. Statistics−Vocabulary and Symbols−art 1:
Laboratory Guide to Method Validation and Related Topics, 2nd. ed.; Probability and General Statistical Terms; International Organization for
Magnusson, B., Ö rnemark, U., Eds.; Eurachem: Teddington, U.K., Standardization: Geneva, Switzerland, 2006.
2014. http://www.eurachem.org (accessed May 28, 2017). (24) Engineered Software, Inc. Repeatability and Reproducibility;
(2) Shabir, G. A. Step-by-Step Analytical Methods and Protocol in Engineered Software, Inc.: Belleville, MI, 1999. http://www.
the Quality System Compliance Industry. J. Valid. Technol. 2004, 10 engineeredsoftware.com/papers/msa_rr.pdf (accessed May 28, 2017).
(4), 314−324. (25) Llamosa, L. R.; Meza, L. G.; Botero, M. Estudio de Repetibilidad
(3) Bolton, S.; Bon, C. Pharmaceutical Statistics, 4th ed.; Marcel y Reproducibilidad Utilizando el Método de Promedios y Rangos para
Dekker: New York, 2004. el Aseguramiento de la Calidad de los Resultados de Calibración de
(4) Riley, C. M.; Rosanske, T. W. Development and Validation of Acuerdo con la Norma Técnica NTC-ISO/IEC 17025. Scientia Et
Analytical Methods; Elsevier: Oxford, U.K., 1996. Technica. 2007, 13 (35), 455−460.
(5) Stitzel, S. E.; Sours, R. E. High-Performance Liquid (26) Montgomery, D. C. Introduction to Statistical Quality Control;
Chromatography Analysis of Single-Origin Chocolates for Methyl- Wiley: New York, 2009; Vol. 7, pp 368−378.
xanthine Composition and Provenance Determination. J. Chem. Educ. (27) Duncan, A. J. Quality Control and Industrial Statistics, 5th ed.;
2013, 90 (9), 1227−1230. Richard D. Irwin: Homewood, IL, 1986.
(6) Usher, K. M.; Simmons, C. R.; Keating, D. W.; Rossi, H. F. (28) Dell Inc. Statistics: Methods and Applications; Dell: Tulsa, OK,
Determination of Niacinamide in Lotions and Creams Using Liquid- 2007. https://support.quest.com/technical-documents/statistics/
Liquid Extraction and High-Performance Liquid Chromatography. J. current/textbook/16#TOPIC-322268 (accessed May 28, 2017).
Chem. Educ. 2015, 92 (5), 907−910. (29) Burdick, R. K.; Larsen, G. A. Confidence Intervals on Measures
(7) Kristian, K. E.; Friedbauer, S.; Kabashi, D.; Ferencz, K. M.; of Variability in R&R Studies. J. Qual. Technol. 1997, 29 (3), 261−273.
Barajas, J. C.; O'Brien, K. A Simplified Digestion Protocol for the (30) Dolezal, K. K.; Burdick, R. K.; Birch, N. J. Analysis of a two-
Analysis of Hg in Fish by Cold Vapor Atomic Absorption factor R & R study with fixed operators. J. Qual. Technol. 1998, 30 (2),
Spectroscopy. J. Chem. Educ. 2015, 92 (4), 698−702. 163−170.
(8) Donais, M. K.; Whissel, G.; Dumas, A.; Golden, K. Analyzing (31) Baur, J. E.; Baur, M. B.; Franz, D. A. The Ultrasonic Soda
Lead Content in Ancient Bronze Coins by Flame Atomic Absorption Fountain: A Dramatic Demonstration of Gas Solubility in Aqueous
Spectroscopy An Archaeometry Laboratory with Nonscience Majors. J. Solutions. J. Chem. Educ. 2006, 83 (4), 577−580.
Chem. Educ. 2009, 86 (3), 343−346. (32) Levy, J. B.; Hornack, F. M.; Levy, M. A. Simple Determination
(9) Homem, V.; Alves, A.; Santos, L. Development and Validation of of Henry’s Law Constant for Carbon Dioxide. J. Chem. Educ. 1987, 64,
a Fast Procedure to Analyze Amoxicillin in River Waters by Direct- 260−261.
Injection LC-MS/MS. J. Chem. Educ. 2014, 91 (11), 1961−1965.
(10) Buck, R. P.; Lindner, E. Peer Reviewed: Tracing the History of
Selective Ion Sensors. Anal. Chem. 2001, 73 (3), 88A−97A.
(11) Severinghaus, J. W.; Astrup, P.; Murray, J. F. Blood Gas Analysis
and Critical Care Medicine. Am. J. Respir. Crit. Care Med. 1998, 157
(4), S114−S122.
(12) Severinghaus, J. W.; Bradley, A. F. Electrodes for Blood pO2 and
pCO2 Determination. J. Appl. Physiol. 1958, 13 (3), 515−520.
(13) Buck, R. P.; Lindner, E. Recommendations for Nomenclature of
Ion-Selective Electrodes (IUPAC Recommendations 1994). Pure Appl.
Chem. 1994, 66 (12), 2527−2536.
(14) Lane, E. E.; Walker, J. F. Clinical Arterial Blood Gas Analysis;
Mosby: St. Louis, 1987.
(15) Cortón, E.; Kocmur, S.; Haim, L.; Galagovsky, L. Potentiometric
Determination of CO2 Concentration in the Gaseous Phase:
Applications in Different Laboratory Activities. J. Chem. Educ. 2000,
77 (9), 1188−1190.
(16) Guilbault, G. G.; Kuan, S. S. Ion selective electrodes and
biosensors based on ISEs. In Biosensors, Fundamentals, and
Applications; Turner, A. P. F., Karube, I., Wilson, G. S., Eds.; Oxford
University: Oxford, 1987; pp 135−152.
(17) Evans, A. Potentiometry and Ion Selective Electrodes; Wiley:
London, U.K., 1987.
(18) Yang, Q.; Long, J.; Zheng, H.; Tian, X.; Lei, D.; Zhu, X.; Guo,
Y.; Xiao, D. Direct Determination of Gas pH and Carbon Dioxide
Concentration with pH Electrodes. J. Chem. Educ. 2014, 91 (4), 593−
596.
(19) Cortón, E.; Haim, L.; Locascio, G.; Galagosky, L.; Kocmur, S.
CO2-Potentiometric Determination and Electrode Construction, a
Hands-on Approach. J. Chem. Educ. 1999, 76 (9), 1253−1255.

F DOI: 10.1021/acs.jchemed.7b00252
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