Analytical Letters

ISSN: 0003-2719 (Print) 1532-236X (Online) Journal homepage: https://www.tandfonline.com/loi/lanl20

Synergistic Effects of Pseudocolor Imaging,

Differentiation, and Square and Logarithmic
Conversion on Accuracy of Quantification of
Chemical Characteristics Using Test Strips and
Similar Products

Ryoichi Doi

To cite this article: Ryoichi Doi (2019): Synergistic Effects of Pseudocolor Imaging, Differentiation,
and Square and Logarithmic Conversion on Accuracy of Quantification of Chemical Characteristics
Using Test Strips and Similar Products, Analytical Letters, DOI: 10.1080/00032719.2018.1556276

To link to this article: https://doi.org/10.1080/00032719.2018.1556276

Published online: 17 Jan 2019.

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ANALYTICAL LETTERS
https://doi.org/10.1080/00032719.2018.1556276

ORIGINAL RESEARCH ARTICLE

Synergistic Effects of Pseudocolor Imaging, Differentiation,

and Square and Logarithmic Conversion on Accuracy of
Quantification of Chemical Characteristics Using Test Strips
and Similar Products
Ryoichi Doi
Faculty of Social-Human Environmentology, Daito Bunka University, Tokyo, Japan

ABSTRACT ARTICLE HISTORY

Test strips and similar products are highly feasible tools for the rapid Received 26 October 2018
and approximate determination of chemical characteristics. Although Accepted 3 December 2018
the application of both the quantitative observation of coloration
KEYWORDS
and regression modeling has recently enabled these products to
Discrete measures to
become quantitative tools, their precision and accuracy may be fur- continuous measures; error
ther improved. In this study, the pseudocolor imaging of the color- statistics; pseudocolor
ation image, derivative spectrophotometry-like differentiation of the imaging; red-green-blue
coloration values, and logarithmic conversion of the raw and deriva- (RGB) color model;
tive values were compared in terms of the precision and accuracy of test strips
the quantitative determination of corrosiveness, glucose, nitrate, and
pH using the products. The best regression models for the determin-
ation were provided by the combination of pseudocolor imaging
and differentiation (nitrate and pH); pseudocolor imaging, differenti-
ation, and square-conversion (corrosiveness); or all of the techniques
(glucose). When compared to the use of the original 10 raw color-
ation variables of red-green-blue, cyan-magenta-yellow-key black,
and L
a
b
color models only, the above combinations improved
the normalized mean absolute error from 14.8% to 3.09% (corrosive-
ness), 6.33% to 3.15% (glucose), 7.46% to 4.56% (nitrate), and 3.22%
to 0.94% (pH). These achievements were largely attributed to the
combination of multiple variables that have non-linear and nonmo-
notonic relationships with the chemical characteristics.

Introduction
Among the various methods and tools employed in the quantitative determination of
chemical characteristics, test strip (St€ urenburg and Junker 2009) and PackTest
(Takahashi, Takaki, and Satta 2012) products are characterized by their high feasibility.
These products are cost-effective, convenient, quick, easy to use, safe, and have minimal
waste. A variety of test strip and PackTest products are currently commercially avail-
able. These products used to be considered qualitative or semi-quantitative analytical
tools because users would refer to a series of standard colors after the product was

CONTACT Ryoichi Doi roird2000@yahoo.com Faculty of Social-Human Environmentology, Daito Bunka University,
1-9-1 Takashimadaira, Itabashi-Ku, Tokyo 175-8571, Japan.
Color versions of one or more of the figures in this article can be found online on at www.tandfonline.com/lanl.
ß 2019 Taylor & Francis Group, LLC
2 R. DOI

utilized by placing it in contact with the sample, which resulted in coloration (Velcarcel,
Cardenas, and Gallego 2000). These days, however, these products can be employed for
the precise and accurate quantitative determination of the chemical characteristics of
samples. In some cases, a quantitative determination of the chemical characteristics is
advantageous (e.g., the critical effects of blood biochemical characteristics at low levels)
(Lamb, MacKenzie, and Stevens 2009). Subtle but significant indicators may be detected
if the performance of these products improves (Haque et al. 2018). Researchers have
been trying to develop methods to enhance the performance of the products. The pre-
cise and accurate quantitative use of test strip and PackTest products is possible
(Hossain et al. 2012). A possible pitfall has been overlooked, however. The most com-
monly used criterion was the coefficient of determination (R2) as an indicator of preci-
sion. A good R2 value does not, however, guarantee high accuracy (Figure 1; Esposito,
Hopfinger, and Madura 2004). Other statistics must also be carefully considered.
Colorimeters have recently become available as tools for the precise reading of the
colors of solid surfaces, including the surface of test strip and PackTest products, and
liquids (Schiavone et al. 2017). Optical scanners and digital cameras can also be
employed to capture images of test strip and PackTest products that display colors.
These optical tools enable reading of the intensity values of the components of color
models, such as the redness in the red-green-blue (RGB) color model. The intensity of
these color components may, however, be suboptimal as measures of specific chemical
characteristics that result in the coloration of test strip and PackTest products. The use
of test strip or PackTest products combined with colorimeters, digital cameras, or other
optical tools to read the intensity of the components of color models is less likely to
identify the optimal color components if compared with a spectrophotometer (Kim-
Pusateri et al. 2009) because of the limited number of color components available for
colorimeters, digital cameras, and similar optical devices. This could be a disadvantage
for test strip and PackTest products if compared to coloration-based measurements
using spectrophotometers or hyperspectral devices.
To cope with this limitation, the pseudocolor imaging of an acquired image (Figure
2; Doi 2014b) may be a way to increase the chance of identifying the optimal measures
for description of the chemical characteristics in question. Once a color digital image is

Figure 1. Precision and accuracy of regression for the estimation of values. The broken oblique lines
indicate perfect matching between actual and estimated values.
 ANALYTICAL LETTERS 3

obtained using a colorimeter or other digital device, the digital image can be converted
using the pseudocolor imaging technique. From the original color image, the pseudo-
color imaging technique generates multiple pseudocolor images that further generate
multiple grayscale images which carry unique information that the original RGB and
other grayscale images do not. After these image-processing procedures, the grayscale
intensity values for the test strip and PackTest pixels can be read as digital numbers
between 0 and 255.
Another way to increase the chance of identifying the optimal measure is the applica-
tion of derivative spectrophotometry (Talsky, Mayring, and Kreuzer 1978). In this study,
to obtain derivative values, the raw grayscale intensity profile was differentiated on ordi-
nally assigned multiple values of chemical characteristics instead of wavelengths.
Logarithmic (Ln) conversion of the raw and derivative values was also expected to
increase the chance of identifying the best measures for describing the observed changes
(Soini, Leussu, and Hallinen 2013). Square conversion of the values was also involved to
identify nonlinear and nonmonotonic increases/decreases in grayscale intensity as
responses to the chemical characteristic to be determined (Doi and
Ranamukhaarachchi 2009).
This study was conducted to minimize errors in the quantitative determination of
chemical characteristics using test strip and PackTest products. In the minimization,
this study compared the effects of pseudocolor imaging, the differentiation of raw gray-
scale intensity profiles on the values of a chemical characteristic, and the logarithmic

Figure 2. Pseudo-color imaging and data-processing procedures employed in this study. Details of
the RGB true color image are shown in Figure 3. The hexagon and the rectangle at the bottom of
the image are Microsoft standard colors and gamut, respectively.
4 R. DOI

and square conversion of the raw and derivative values on identifying the most accurate
measure for the quantitative determination of a chemical characteristic. Test strip prod-
ucts for the qualitative detection of copper corrosiveness, urinary glucose, and environ-
mental nitrate were investigated in addition to the PackTest product for the
approximate and quick estimation of pH. Images of colored test strip and PackTest
products indicating responses to ranges of the chemical characteristics were obtained.
Through pseudocolor imaging of the acquired color image, 83 grayscale images were
obtained (Figure 2). The grayscale intensities of the grayscale images were read. Eighty-
three derivative values were obtained by differentiating the raw grayscale intensity val-
ues along the gradient of corrosiveness, pH, or concentration. Six datasets were pre-
pared based on the raw, derivative, and Ln- and square-converted raw/derivative values.
The efficacy of the datasets in identifying the most reliable, precise, and accurate regres-
sion model to describe changes in the chemical characteristic was compared to identify
the most effective technique among pseudocolor imaging, differentiation, and Ln and
square conversion. In the identification processes, some error statistics were referenced.
This is the first study to apply the principle of derivative spectrophotometry to chemical
quantification using test strip and PackTest products.

Materials and methods

Test strip and PackTest products
In this study, three test strips and a single PackTest were investigated. For corrosiveness,
a copper test strip product from ASTM International (Pennsylvania, USA) was investi-
gated. For glucose determination, a New Uri-Ace Ga test strip (Terumo Corporation,
Tokyo, Japan) was used (Doi 2014a). A test strip product for nitrate determination
(Quantofix, Macgerey-Nagel, D€ uren, Germany) was also used. For pH, a PackTest from
Kyoritsu Chemical-Check Lab. Corp. (Tokyo, Japan) was investigated.

Loading standard solutions of glucose and nitrate on test strips

Standard solutions of glucose were prepared by dissolving the solid in distilled water.
Eleven standard solutions were prepared. The concentrations were 0.00, 0.11, 0.22, 0.33,
0.44, 0.55, 0.66, 0.77, 0.88, 1.00, and 1.11 mM.
Potassium nitrate was dissolved in distilled water to prepare standard solutions of
0.00, 0.16, 0.32, 0.48, 0.65, 0.81, 0.97, 1.13, 1.29, 1.45, and 1.61 mM. The standard solu-
tions were applied to the pad of the test strip at four micro L pad1 (glucose) or ten
micro L pad1 (nitrate). The test strip loaded with the sample solution was incubated
for 3 hours (glucose) or 2 hours (nitrate) in the dark at room temperature (around
25  C) (Doi 2014a). To avoid desiccation, the test strips were kept in a plastic box with
water on the bottom.

Image acquisition and processing

The rectangular pad of the glucose or nitrate test strip was collected using an Epson
GT-S 650 optical scanner. The test strip was scanned at 600 dots per inch in
 ANALYTICAL LETTERS 5

professional mode. Images of the test strips were saved as JPG files, and then converted
to tag image file format files in RGB mode with the sRGB color space. Images of the
standard colors for the copper corrosiveness test strip and the pH PackTest product
were obtained from the Internet. For the copper corrosiveness test strip, images of
standard colors in the instructions were downloaded from a distributor’s website
(https://www.refinerysupply.com). For the pH PackTest product, the standard color
images in the instructions were downloaded from the manufacturer’s website (http://
kyoritsu-lab.co.jp/english/). The standard colors represent pH values of 2 to 13. For the
downloaded images of the standard colors for corrosiveness and pH, the image datum
was pasted into a file window of Adobe Photoshop 7.0 in RGB mode applying the
sRGB color space. Images of the corrosiveness and pH standard colors were saved as
tag image file format files.
Using the Adobe Photoshop software, each of the tag image file format images gener-
ated 10 grayscale tag image file format images that indicated the grayscale intensity val-
ues for the color components of RGB, cyan-magenta-yellow-key black (CMYK), and the
International Commission and Illumination’s L
a
b
color models. CMYK images were
generated with the International Color Consortium profile of US Web Coated (SWOP)
v2 for digital output such as color printing. An RGB yellow grayscale image was pre-
pared by merging the R and G grayscale images at the same weights (Doi 2012).
Likewise, RGB cyan and RGB magenta grayscale images were prepared by merging the
G and B grayscale images and the R and B grayscale images, respectively.
To prepare a pseudocolor RGB image, the entire area of a B grayscale image of an
RGB color image was substituted by the RGB yellow grayscale image. Hereafter, this
image is called the RGyB image (Figure 2). A pseudocolor RG-yB image was prepared
by substituting the B grayscale image of the RGB color image with the black-white
inverted RGB yellow grayscale image. Likewise, an RmGB pseudocolor image was pre-
pared by putting the RGB magenta grayscale image on the entire area of a G grayscale
image of an RGB image. R-mGB, cRGB, and -cRGB pseudocolor images were also pre-
pared and saved.
Pseudocolor images carrying two or three of the RGB cyan, RGB magenta, and RGB
yellow grayscale images were also prepared (Figure 2). For example, the G and B gray-
scale images of an RGB image were substituted with the RGB magenta and yellow
images, respectively. Hereafter, this image is called the RmGyB pseudocolor image.
Likewise, cRGyB and cRmGB pseudocolor images were prepared. By substituting the R,
G, and B grayscale images of the RGB color image with the RGB cyan, magenta, and
yellow hybrid grayscale images, respectively, a cRmGyB pseudocolor image was
also prepared.
The 10 pseudocolor images were saved as tag image file format files. Each of the
pseudocolor images was converted to CMYK and L
a
b
color images. Next, grayscale
images that showed the intensity values of C, M, Y, K, and L
and the values of a
and
b
were prepared from each pseudocolor image (Figure 1). Hereafter, the C, M, Y, K,
L
, a
, and b
images are referred to as the RGyB C grayscale image and so on. Thus,
70 grayscale images were added to the original 10 grayscale images and the three hybrid
grayscale images of RGB yellow, RGB magenta, and RGB cyan. A total of 83 grayscale
images were obtained for each chemical characteristic.
6 R. DOI

Datasets for multiple regression

The grayscale intensity values for pixels representing the standard colors for the pH val-
ues and those representing the colored test strip surface were read by running
MultiSpec version 3.4 for Windows (Purdue Research Foundation, USA). The intensity
values were digital numbers between 0 (complete black) and 255 (complete white).
Within a color image for a corrosion severity value, a glucose/nitrate concentration, or
a single pH value, eight replication pixels were randomly selected. The grayscale inten-
sity values for the selected pixels were read for the 83 grayscale images.
For glucose, a series of raw grayscale intensity values for the 11 pixels representing
the 11 standard glucose concentrations was handled as a raw grayscale intensity pro-
file. A second derivative profile on the range of glucose concentrations was obtained
by sacrificing the values of the smallest and largest glucose concentrations. For each
grayscale image, the second derivative profile was obtained for the series of nine pix-
els/concentrations. Thus, each grayscale image had eight raw and eight derivative repli-
cation profiles. Second derivative profiles for nitrate and pH were obtained in the
same manner. In the case of corrosiveness, ordinal values were given to obtain a con-
tinuous quantitative measure representing corrosiveness classes for newly prepared
copper surfaces and those representing 12 severity levels of copper corrosion (ASTM
International 2010). Based on the ordinal values, a derivative corrosiveness profile for
each grayscale image was obtained. Thus, 83 raw and 83 derivative variables occurred
for eight pixel replications  nine levels (glucose and nitrate), 10 levels (pH), or 11 lev-
els (corrosiveness).
Based on the raw and derivative values, six datasets were constructed (Figure 1). The
first dataset consisted of grayscale intensity values for the original 10 grayscale images
for the intensity values of RGB, CMYK, and L
a
b
color components. The number of
variables of this dataset was 10. Hereafter, this dataset is referred to as the original
10 dataset.
The second dataset included the raw intensity values for the original 10, the three
hybrid grayscale, and the 70 grayscale images from the 10 pseudocolor images (Figure
1). Thus, this dataset consisted of 83 variables. In this manuscript, this dataset is called
the 83 images’ dataset.
The third was the 83 þ d-83 dataset. This dataset consisted of the 83 variables and
the second derivative values for the 83 grayscale images. This dataset, therefore, had
166 variables.
The fourth was the 166 þ sq-166 dataset. To prepare this dataset, the 83 raw and 83
derivative values were squared and then combined with the 83 þ d-83 dataset. This gen-
erated 332 variables.
The fifth was the 166 þ Ln-166 dataset. This dataset was prepared by Ln converting
the 83 þ d-83 dataset and combining it with the 83 þ d-83 dataset. Before the Ln con-
version of the derivative values, the derivative values were squared to avoid negative
derivative values that disabled Ln conversion. These procedures provided 332 variables
to the 166 þ Ln-166 dataset.
The sixth dataset had 498 variables and is referred to herein as the all-in-one dataset.
This dataset is the combination of the 332 variables of the 166 þ sq-166 dataset and the
Ln-converted 166 variables. These datasets were compared to identify the most effective
 ANALYTICAL LETTERS 7

dataset in the quantitative determination of the four chemical characteristics in terms

of accuracy.

Statistical analyses
Using the SPSS statistical software 10.0.1 (Illinois, USA), multiple regression analysis
was performed to examine the significant relationships between the values of the varia-
bles and the values of chemical characteristics. In the computation, the stepwise method
with the default criteria (p = 0.05 for inclusion and 0.10 for removal) was chosen. The
statistical analysis estimated the coefficients and a constant to describe the sample’s
chemical characteristics.
Value of biochemical characteristic
¼ a  value of variable 1 þ b  value of variable 2 þ       þ constant
(1)
Here, a, b, and others are coefficients. The analysis provides t-statistics for the coeffi-
cients and constant. Thus, the reliability of the estimated coefficients and constant is
quantified. In addition to these reliability statistics, through the above statistical analy-
ses, the normalized mean absolute error (Yoshizawa et al. 1981) and coefficient of deter-
mination (R2) were obtained as indicators of accuracy and precision, respectively. The
normalized mean absolute error was determined by

P Normalized mean absolute error ð%Þ

¼ 100  ð jEstimated value – actual valuejÞ=number of used pixels=range of value
(2)
The values of these statistics were compared among the best regression models for
the six datasets to investigate the effects of pseudocolor imaging, differentiation of color
profile along the gradient of corrosiveness, concentration, or pH, and Ln and square
conversion of the raw and derivative values on the reliability, precision, and accuracy in
the determination of the chemical characteristics.
To confirm the performance of the best regression model obtained in the above proc-
esses, the coefficient of variation as another precision statistic based on the eight repli-
cations was determined by
Coefficient of variation (%) = 100  standard deviation/mean (3)
To observe the data structures of the datasets used in this study, values of the 498
variables  39 (11 þ 9 þ 9 þ 10) levels  eight replication pixels were used for principal
component analysis using SPSS software. The simple linear relationship between the sig-
nificant variable for the multiple regression and the chemical characteristic was also
examined by SPSS software.

Results and discussion

Figure 3 indicates the color changes of the test strip and PackTest products as
responses to corrosion severity, glucose and nitrate concentrations, and pH. Corrosion
severity and pH are shown as complicated patterns of changes. In contrast, glucose
8 R. DOI

and nitrate test strips showed simpler changes in color. Greater concentrations of glu-
cose resulted in dark green colors. As nitrate was loaded at great concentrations, the
pad became pink. Although the glucose concentrations are far below critical as urinary
glucose concentrations, glucose measurement can be applicable to the quantification of
glucose in other samples, such as soils (Doi 2014a). Nitrate in drinking water should
not exceed 0.67 mM (Tang et al. 2004). Hence, the quantitative determination of glu-
cose and nitrate at these concentrations is useful, even though the reaction time
adopted in this study was much longer than the 30 seconds (glucose) and 1 minute
(nitrate) recommended in the instructions. This extension of the reaction time enables
sensitive determination of these compounds at lower ranges (Figure 3) than between
2.78 and 111 mM (glucose) and between 0.16 and 8.06 mM (nitrate) described in the
instructions.
Table 1 presents the results of the multiple regression analysis to identify significant
variables for the determination of chemical characteristics. Regression models that
include coefficients with variance inflation factors of 10 or greater were eliminated
because they are badly affected by multicollinearity effects (O’brien 2007). For corrosive-
ness, a regression model was found to include a variable with a p value larger than 0.05.
The regression model was also eliminated in the following processes. All datasets gener-
ated significant regression models for description of the chemical characteristics. In
most regression models, the chemical characteristics were described by multiple varia-
bles. When the original 10 dataset was supplemented with the derivative and/or con-
verted variables, a tendency of greater R2 and smaller normalized mean absolute error
was recognized. For example, pH was best determined by the 83 þ d-83 dataset, with
which, the R2 and normalized mean absolute error values improved from 0.985 to 0.998
and 3.22% to 0.94%, respectively. Similar effects were observed in the determination of
corrosiveness, glucose, and nitrate, which were best determined by the 166 þ sq-166, all-
in-one, and 83 þ d-83 datasets, respectively. The values of the normalized mean absolute
error listed in Table 1 indicate that the best dataset for quantitative determination
depends on the chemical characteristics to be determined. A larger number of variables
does not always provide the best regression model because of collinearity effects that
result in variables with large variance inflation factors and exclude the models from the
following steps.
In describing corrosiveness, nitrate, and pH, the addition of the derivative variables
to the 83 images’ dataset was effective, as indicated by the values of normalized mean
absolute error that largely dropped from those for the original 10 and the 83 images’
dataset. Among the four chemical characteristics, the accuracy of corrosiveness deter-
mination relying on the color profiles of copper surface was most markedly improved
when the 83 derivative variables were added (Table 1). When the original 10 variables
were used for multiple regression analysis, the accuracy was very poor (Figure 4).
Reading the intensity values for the original 10 color components could not successfully
discriminate between 1a or 1 b and 3a (Figures 3 and 4), which have some appearance
properties in common, and are thus difficult to discriminate between and may be mis-
recognized (ASTM International 2010). This problem was solved by the regression
model derived from the 83 þ d-83 dataset. It enabled clear discrimination between the
similar appearance properties. Copper and copper alloys are relatively more corrosion-
Table 1. Multiple regression statistics for grayscale images forming the most significant regression models for the sets of grayscale images used for deter-
mination of the chemical characteristics.
Constant or coefficient Normalized mean
Chemical characteristic Significant Significance Variance Coefficient of absolute error
to be determined Data set grayscale imagea Value 　t (p value)　 inflation factor determination (%)
Corrosive Original 10 Constant 43.1 13.4 <0.001 — 0.616 14.8
1 to 11, ordinal value 　 K –0.114 –7.32 <0.001 1.41
　 a
–6.53  10-2 –3.93 <0.001 1.41
83 images Constant 11.2 21.4 <0.001 — 0.576 16.6
　 RG-yB C –3.34  10-2 –10.2 <0.001 1.00
83 þ d-83 Constant 28.8 50.8 <0.001 — 0.972 3.99
　 RGyB a
–0.101 –17.3 <0.001 3.74
　 d RGyB a
–0.219 –25.6 <0.001 2.85
dB –0.155 –13.5 <0.001 4.32
　 cRGB Y –5.10  10-2 –11.2 <0.001 6.52
　 d RGyB C 2.43  10-2 5.89 <0.001 6.98
166 þ sq-166 Constant 9.18 13.6 <0.001 — 0.983 3.09
sqd Y –8.26  10–4 –4.29 <0.001 7.65
　 sqd a
–7.09  10-3 –9.83 <0.001 4.42
　 sqd R-mGB K 3.64  10-3 7.14 <0.001 2.18
　 d cRGyB b
0.159 15.2 <0.001 4.81
　 sq RGyB a
–2.64  10-4 –12.9 <0.001 6.83
　 d RmGB Y –6.55  10-2 –8.45 <0.001 4.77
　 R-mGB b
2.69  10-2 4.91 <0.001 2.37
166 þ Ln-166 Constant –267 –20.0 <0.001 — 0.982 3.19
Ln RGyB b
38.2 25.0 <0.001 7.23
　 d a
–0.187 –20.0 <0.001 4.26
　 Ln cRGyB K 17.4 13.4 <0.001 3.67
　 Ln(d RGyB b
)2 0.237 5.21 <0.001 2.75
　 Ln(d R-mGB C)2 0.153 4.99 <0.001 3.81
　 Ln(d RmGB K)2 0.415 15.6 <0.001 7.88
　 Ln(d cRGyB C)2 0.159 3.39 0.001 2.08
　 Ln(d cRGyB Y)2 –9.10  10-2 –3.94 <0.001 8.32
　 Ln(d cRmGB L
)2 0.243 5.37 <0.001 6.50
All-in-one Constant –267 –20.0 <0.001 — 0.982 3.19
Ln RGyB b
38.2 25.0 <0.001 7.23
d a
–0.187 –20.0 <0.001 4.26
　 Ln cRGyB K 17.4 13.4 <0.001 3.67
ANALYTICAL LETTERS

　 Ln(d RGyB b
)2 0.237 5.21 <0.001 2.75

　 Ln(d R-mGB C)2 0.153 4.99 <0.001 3.81
(continued)
9
 10

Table 1. Continued.
Constant or coefficient Normalized mean
Chemical characteristic Significant Significance Variance Coefficient of absolute error
to be determined Data set grayscale imagea Value 　t (p value)　 inflation factor determination (%)
Ln(d RmGB K)2 0.415 15.5 7.88
R. DOI

　 <0.001
　 Ln(d cRGyB C)2 0.159 3.39 0.001 2.08
　 Ln(d cRGyB Y)2 –0.10  10-2 –3.94 <0.001 8.32
　 Ln(d cRmGB L
)2 0.243 5.37 <0.001 6.50

Glucose Original 10 Constant 1.29 12.1 <0.001 — 0.944 6.33

(mM) 　 K –5.49  10-3 –12.7 <0.001 3.13
　 Y 8.38  10-3 7.77 <0.001 3.13
83 images’ Constant 1.00 16.8 <0.001 　 0.945 6.17
　 RG-yB M –5.79  10-3 –30.5 <0.001 1.16
　 cRmGB Y 1.04  10-2 4.83 <0.001 1.16
83 þ d-83 Constant 0.859 9.99 <0.001 — 0.958 5.56
　 RG-yB M –4.31  10-3 –13.6 <0.001 4.22
　 d RmGyB a
4.96  10-2 4.79 <0.001 1.63
　 cRGB Y 6.64  10-3 4.66 <0.001 4.06
166 þ sq-166 Constant 1.09 32.4 <0.001 — 0.965 4.79
RG-yB M –5.05  10-3 –21.3 <0.001 2.81
　 sq cRmGB Y 2.14  10-4 5.28 <0.001 1.22
　 d RmGyB a
7.83  10-2 5.81 <0.001 3.28
　 sqd RmGyB a
2.24  10-2 3.52 0.001 3.09
　 sqd R-mGB K –1.66  10-4 –2.17 0.033 2.54
166 þ Ln-166 Constant 0.754 10.8 <0.001 — 0.979 3.56
RG-yB M –2.87  10-3 –8.35 <0.001 9.91
　 d RmGyB a
5.74  10-2 5.75 <0.001 3.03
　 cRGB Y 6.13  10-3 5.47 <0.001 5.02
　 Ln(d RmGyB a
)2 1.46  10-2 3.63 0.001 2.25
　 Ln(d R-mGB C)2 –8.25  10-3 –2.04 0.046 8.91
　 Ln(d RG-yB K)2 1.07  10-2 4.42 <0.001 2.00
　 Ln(d RmGB C)2 –2.33  10-2 –4.37 <0.001 9.34
　 Ln(d RGyB K)2 2.49  10-2 5.22 <0.001 4.97
　 d RmGB a
–4.26  10-2 –4.23 <0.001 4.92
　 Ln(d RG-yB a
)2 –9.58  10-3 –2.58 0.012 1.99
All-in-one Constant 1.00 30.8 <0.001 — 0.983 3.15
RG-yB M –3.86  10-3 –14.7 <0.001 7.01
sq cRmGB Y 1.72  10-4 5.67 <0.001 1.38
　 d RmGyB a
4.21  10-2 3.52 0.001 5.23
 　 sqd RmGyB a
1.79  10-2 3.18 0.002 4.87
　 Ln(d R-mGB C)2 –1.59  10-2 –4.18 <0.001 9.46
　 Ln(d RGyB K)2 2.51  10-2 6.56 <0.001 3.84
　 Ln(d cRmGyB Y)2 –1.91  10-2 –3.70 <0.001 4.01
　 sqd RG-yB K 2.98  10-4 6.40 <0.001 3.44
　 d cRGyB K 4.72  10-3 4.32 <0.001 2.55
　 Ln(d cRGB C)2 –1.64  10-2 –3.90 <0.001 3.99
Nitrate Original 10 Constant 7.68 8.74 <0.001 — 0.917 7.46
(mM) 　 M –2.78  10-2 –27.4 <0.001 1.39
　 C 4.87  10-2 8.79 <0.001 2.96
　 Y –5.49  10-2 –8.17 <0.001 2.79
83 images’ Constant –88.4 –7.13 <0.001 — 0.914 9.01
　 RmGB b
0.176 19.4 <0.001 2.68
　 cRmGB M 0.125 4.88 <0.001 1.09
　 RG-yB K 0.123 3.30 0.002 2.61
83 þ d-83 Constant –11.7 –2.20 0.031 — 0.968 4.56
　 R-mGB C 2.97  10-2 9.29 <0.001 2.44
　 RmGB Y –2.76  10-2 –16.9 <0.001 2.31
　 d -cRGB Y 2.39  10-2 9.12 <0.001 1.89
　 d RGyB a
3.40  10-2 4.24 <0.001 1.53
　 cRmGB M 7.25  10-2 3.23 0.002 1.99
　 cRmGyB b
–4.99  10-2 –2.67 0.010 1.23
166 þ sq-166 Constant –18.9 –20.1 <0.001 — 0.945 6.66
RmGB b
0.113 15.4 <0.001 2.44
　 d cRGB b
–9.42  10-2 –6.49 <0.001 1.86
　 R-mGB C 1.62  10-2 4.35 <0.001 1.93
166 þ Ln-166 Constant –94.2 –20.2 <0.001 — 0.953 5.60
Ln RmGB b
16.2 15.4 <0.001 2.58
　 d cRGB b
–7.56  10-2 –5.19 <0.001 2.15
　 Ln R-mGB C 2.74 4.57 <0.001 1.92
　 Ln(d b
)2 2.01  10-2 3.13 0.003 1.80
All-in-one Constant –98.7 –20.9 <0.001 — 0.947 6.00
Ln RmGB b
17.0 15.6 <0.001 2.43
d cRGB b
–9.21  10-2 –6.39 <0.001 1.87
　 Ln R-mGB C 2.85 4.48 <0.001 1.91

pH Original 10 Constant 24.3 38.8 <0.001 — 0.985 3.22

a
–7.21  10-2 –40.0 <0.001 2.30
B –3.09  10-2 –9.60 <0.001 3.02
ANALYTICAL LETTERS

b
–1.64  10-2 5.40 <0.001 1.49

83 images’ Constant 25.0 38.5 <0.001 — 0.984 3.38
(continued)
11
 12

Table 1. Continued.
Constant or coefficient Normalized mean
Chemical characteristic Significant Significance Variance Coefficient of absolute error
to be determined Data set grayscale imagea Value 　t (p value)　 inflation factor determination (%)
1.04
R. DOI

a
–8.26  10-2 –67.2 <0.001

cRGB C –2.91  10-2 –9.25 <0.001 1.04
83 þ d-83 Constant 25.3 104 <0.001 — 0.998 0.94
a
–7.70  10-2 –156 <0.001 1.58
cRGB C –1.82  10-2 –15.8 <0.001 1.31
d RmGB Y –1.90  10-2 –18.2 <0.001 1.66
RG-yB K –1.48  10-2 –15.9 <0.001 1.34
d RmGyB K 0.150 12.2 <0.001 1.12
166 þ sq-166 Constant 25.1 38.5 <0.001 — 0.984 3.38
a
–8.26  10-2 –67.2 <0.001 1.04
cRGB C –2.91  10-2 –9.25 <0.001 1.04
166 þ Ln-166 Constant 16.8 97.8 <0.001 — 0.994 2.06
a
–7.55  10-2 –74.3 <0.001 1.85
Ln(d RmGyB Y)2 0.177 10.6 <0.001 1.34
Ln(d cRGyB Y)2 7.24  10-2 9.02 <0.001 2.28
All-in-one Constant 18.8 182 <0.001 — 0.995 1.86
a
–8.42  10-2 –120 <0.001 1.00
sqd cRmGyB Y 4.91  10-4 20.1 <0.001 1.00
a
The variable with d followed by a grayscale image name indicates a derivative variable, and that with sq and Ln a square- and a logarithmic-converted variable, respectively.
 ANALYTICAL LETTERS 13

resistant than iron and some other metals (Nagata 1987). In some conditions, however,
corrosion occurs. The severity differs depending on conditions such as the concentra-
tion of sulfur dioxide in petroleum products. The current method to combine pseudo-
color imaging, differentiation, and conversion would be beneficial to sensitively detect
the corrosiveness of petroleum products (Andersen, Abdulagatov, and Bruno 2003), the
atmospheric environment (Watanabe et al. 2006), and other samples.
R2 is an indicator of precision, but a great value of R2 does not always guarantee
good accuracy (Kenyeres et al. 2011). Care must be taken when selecting the best
regression model for the quantitative determination of a chemical characteristic. If
regression models with great R2 values have insignificant coefficients (Bernardo 2018)
and constants (El-Khaiary and Malash 2011), the regression is unreliable and inaccurate,
even if the R2 values are large. For example, as shown in the bottom right diagram in
Figure 1, if a constant is unreliable, the regression model is unreliable and inaccurate,
even if the R2 value is large. To exclude such unreliable and inaccurate regression mod-
els, it is effective to rely on a relevant value of variance inflation factor as a threshold in
addition to suitable inclusion-exclusion criteria. R2 values merely indicate the precision
of regression models. R2 values cannot be the sole decisive indicators of the reliability
and accuracy of regression models. The statistics referred to in this study (Table 1) help
users identify the most reliable, precise, and accurate measure that consists of a constant
and coefficients which have seldom been considered in the determination of the chem-
ical characteristics of samples relying on regression models (Megargle, Jones, and
Rosenthal 1970; Ellison, Holcombe, and Burns 2001; Srbek et al. 2003).
In this study, eight replication pixels were used for each value of the chemical charac-
teristics. When coefficients of variation were calculated for the greatest values of corro-
siveness, concentrations, and pH with the best regression models, the values were 3.07%
(corrosiveness, 11 as C4b), 3.37% (glucose, 1.00 mM), 4.84% (nitrate, 1.45 mM), and
0.92% (pH 12). These values are comparable to those of normalized mean absolute error
in Table 1. This is because approximately 67% [Eq. (3), Moerman 1979] and 50% of the
observed values should fall within the ranges of the coefficient of variation and normal-
ized mean absolute error, respectively. These values are commonly acceptable according
to the widely applied criteria in the quantitative determination of chemical characteris-
tics as well as the number of replication pixels used for each value of corrosiveness, pH,
or concentration (Shah et al. 1992). These values of the coefficient of variation are close
to those of the well-established methods for quantitative chemical determination, such
as 3.3% for the colorimetrical determination of diazepam with 11 replications
(Liawruangrath, Makchit, and Liawruangrath 2006), and 4 to 10% for heavy metals in
grapevine leaves when 10 replications were supplemented for their determination by
inductively coupled plasma–mass spectrometry (Bokowski et al. 2016).
The importance of replications is suggested in Figure 4. Shah et al. (1992) recom-
mended five or greater replications for each quantitative standard level, and also recom-
mended the setting of a suitable number and a range of standard levels. The diagram of
glucose coloration (Figure 3) suggests that the greatest concentration of 1.0 mM glucose
applied in this study was around the maximum for this method, which can detect as
low as 0.005 mM glucose (Doi 2014a). This indicates the importance of preparing stand-
ard levels covering a suitable range of concentrations. Dilution of samples may also be
14 R. DOI

Figure 3. Standard colors used in this study. Classification (
) indicates the series of classes of copper
corrosion severity described in the manufacturer’s instructions.

Figure 4. Effect of pseudo-color imaging, differentiation of the raw grayscale intensity profile, and
square conversion of the raw and derivative values on the precision of the determination of corrosive-
ness by using the copper test strip. The red-colored oblique lines indicate perfect matching between
actual and estimated values.

helpful for the precise and accurate determination of chemical characteristics using test
strip and PackTest products, pseudocolor imaging, current techniques for dataset prep-
aration, and regression analysis.
By comparing the improvement in the values of R2 and normalized mean absolute
error as a result of the addition of derivative values as well as Ln- and square-converted
values, there appear to be synergistic effects among the variables in addition to the
uniqueness of the groups of variables (Table 1). The results of principal component
 ANALYTICAL LETTERS 15

analysis elucidate the synergistic effects of the groups of variables that differed uniquely
from one another (Figure 5). According to Figure 5, the variables of the 83 images’
dataset tended to have large loadings on the first principal component whereas those of
the 83 derivative variables tended to have large loadings on the second principal compo-
nent. Many of the Ln-converted variables had unique patterns of loadings on the most
significant principal components, as in the case of the best regression model for glucose
determination (Table 1).
As depicted in Figure 6, many among the significant variables of the best regression
models for the chemical characteristics in Table 1 had poor linear relationships (R2 <
0.5) with the chemical characteristics. Among them, the most significant linear relation-
ship with a chemical characteristic was observed for a
in the regression model for pH
(Table 1), indicated by the PackTest appearance properties between reddish and green-
ish colors (Figure 3) that should closely relate to the value of a
(Figure 2; Lakio,
Hein€am€aki, and Yliruusi 2010). The R2 value for the simple linear regression was only
0.968, however (Figure 6). The regression model derived from the 83 þ d-83 dataset
consisted of five variables, including a
(Table 1). The other four variables were less lin-
early associated with pH (Figure 6) whereas the combination increased the R2 value to
0.998 and achieved a very narrow normalized mean absolute error of 0.94% (Table 1).
This improvement in accuracy was clearly attributed to the combination of the five vari-
ables, among which, four had less linear relationships with pH than a
.


3ULQFLSDOFRPSRQHQW



3ULQFLSDOFRPSRQHQW

LPDJHVಬGVT(LQ)LJ/Q)LQ)LJ

Figure 5. Loadings for the groups of variables on the first and second principal components
extracted from the values for the pixels in the 39 standard colors among the 47 in Figure 3.
16 R. DOI

.K
Da*
R-mGBE
5P*% b*
G5P*%<
d RmGB Y &RUURVLYHQHVV
d cRGyB E
GF5*\% b*
sq RGyB D
VT5*\% a*
VTG<
sqd Y
sqd D
VTG a*
VTG5P*%.
sqd R-mGB K

<
Y
.
K
5*\%0
RG-yB M
G5P*\%D
d RmGyB a*
GF5*\%.
d cRGyB K
VTF5P*%<
sq cRmGB Y *OXFRVH
VTG5*\%.
sqd RG-yB K
sqd RmGyBDa*
VTG5P*\%
/QG5*\%.
Ln(d RGyB K)2 

/QG5P*%&
Ln(d R-mGB C)2 

/QGF5*%&
Ln(d cRGB C)2 

/QGF5P*\%<
Ln(d cRmGyB Y)2 

&C
M
0
Y
<
RmGB Y
5P*%<
R-mGB C
5P*%&
cRmGB
F5P*%0 M
cRmGyB E
F5P*\% b*
d RGyBDa*
G5*\%
1LWUDWH
d -cRGB Y
GF5*%<
B
%
Da*
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RG-yB K
5*\%.
cRGB C
F5*%&
d RmGyB K
S+
G5P*\%.
d RmGB Y
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&RHIILFLHQWRIGHWHUPLQDWLRQR2

Figure 6. Simple linear relationships between the significant variables listed in Table 1 and the chem-
ical characteristics. The variable with d followed by a grayscale image name indicates a derivative
variable, and those with sq and Ln a square- and a logarithmic-converted variable, respectively.

The combination of variables having nonlinear relationships with a chemical charac-

teristic to be measured is thus worth trying. Based on measurements of color, it appears
to be difficult to identify a single variable for the reliable, precise, and accurate quantita-
tive determination of the chemical characteristic. For this reason, a realistic way is to
obtain a measure by combining multiple variables that have complicated patterns
of changes.
Figure 7 shows how the significant variables of the best regression model for corro-
siveness are associated with corrosion severity values. None of the variables had an even
monotonic increase/decrease relationship to corrosion severity. As seen in the above
examples, the combination of nonlinear variables may provide a reliable, precise, and
accurate measure for the quantitative determination of chemical characteristics.
Pseudocolor imaging generated minor but significantly effective information for dis-
crimination among similar agricultural plots (Doi 2016b) and forest vegetation types
(Doi 2016a). In contrast, the improvement of precision and accuracy achieved in this
study was only partially supported by pseudocolor imaging. The synergistic effects of
the combination of pseudocolor imaging, the differentiation of raw grayscale intensity
values, and Ln and square conversion were more effective (Table 1, Figures 6 and 7).
This combination was realized by the variables that uniquely differ and are independent
from one another. Thus, searching for the best variables from them should increase the
chance to identify a new regression model that improves the accuracy of the quantita-
tive determination of the chemical characteristic.
 ANALYTICAL LETTERS 17


VT5*\%D



VTG<



VTG5P*%.
VTGD



GF5*\%E 
G5P*%<



.
D
5P*% E 



&RUURVLRQVHYHULW\RUGLQDOYDOXH

Figure 7. Nonlinear and nonmonotonic relationships between corrosion severity and values of the
significant variables of the best regression model (166 þ sq-166 dataset) and those relying on the ori-
ginal 10 variables (K and a
) for corrosiveness. The variables with d and sq(d) followed by a grayscale
image name indicate a derivative and a square-converted (derivative) variable, respectively.

Test strip and PackTest products are highly useful for the determination of the chem-
ical characteristics of samples. These products are currently becoming more accurate,
precise, and quantitative, largely owing to the development of color-reading tools, such
as scanners (Kostenko 2009) and digital processing (Yetisen, Akram, and Lowe 2013) of
the acquired image data. The products are commercially available for diagnosis (Pfab
et al. 2006), the analysis of pesticides in foods (Hossain et al. 2009), infectious microbes
(Blazkova et al. 2011), and others. In the quantitative determination of chemical charac-
teristics involving derivative spectrophotometry-like differentiation, Ln and square con-
version of raw and derivative values, and pseudocolor imaging, test strip and PackTest
products and similar color-/spectrum-based products can be considered reliable, precise,
and accurate tools that are useful in laboratories and hospitals (Delanghe et al. 2017),
schools (Rahman 2011), and even ordinary homes (Doi 2014a).
18 R. DOI

Conclusions
For the reliable, precise, and accurate quantitative determination of corrosiveness, glu-
cose, nitrate, and pH using test strip and PackTest products, pseudocolor imaging, dif-
ferentiation, and Ln and square conversion exhibited synergistic effects. The groups of
variables showed uniqueness and independence from one another. These unique pat-
terns were advantageous when it was very difficult to identify a single variable for the
quantitative determination of the chemical characteristic because a helpful regression
model is generated by combining multiple variables that have uniquely different pat-
terns to the gradient of the chemical characteristic. Taking the advantages observed in
this study into consideration is useful when using test strip and PackTest products and
similar tools that rely on coloration to represent the chemical characteristics to be deter-
mined. In this way, these highly feasible products can become reliable, precise, and
accurate quantification tools with the aid of statistics that critically select the best meas-
ures among those generated by statistical regression analyses. Once an RGB, CMYK, or
any other true color image is obtained, then users can easily prepare datasets to identify
the best measure for the accurate determination of the chemical characteristic. The pro-
cedures examined in this study may be applicable to various quantitative determinations
of chemical and other characteristics of samples.

Disclosure statement
The author declares no conflicts of interest regarding this manuscript.

Funding
This study was conducted using the budget of our institute.

ORCID
Ryoichi Doi http://orcid.org/0000-0002-3023-0136

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