Trends in Analytical Chemistry 138 (2021) 116223

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Trends in Analytical Chemistry

journal homepage: www.elsevier.com/locate/trac

White Analytical Chemistry: An approach to reconcile the principles of

Green Analytical Chemistry and functionality
Paweł Mateusz Nowak a, Renata Wietecha-Posłuszny a, *, Janusz Pawliszyn b
a
w, 2 Gronostajowa St., 30-
Laboratory for Forensic Chemistry, Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University in Krako

w, Poland
387, Krako
b
Department of Chemistry, University of Waterloo, 200 University Avenue West, Ontario, N2L 3G1, Canada

a r t i c l e i n f o a b s t r a c t

Article history: The concept of White Analytical Chemistry (WAC) is presented as an extension of Green Analytical
Available online 16 February 2021 Chemistry (GAC). We propose the 12 WAC principles as an alternative to the known 12 GAC principles. In
addition to green aspects, WAC takes into account other key criteria affecting the quality of the method,
Keywords: analytical (red) and practical (blue). In reference to the RGB color model, according to which mixing of
Analytical method red, green and blue light beams gives the impression of whiteness, a white analytical method shows the
Assessment tool
coherence and synergy of the analytical, ecological and practical attributes. Whiteness can also be
Green analytical chemistry
quantified, based on the assessment of individual principles, as a convenient parameter useful in com-
RGB model
Sample preparation
parisons and selecting optimal method. WAC is closer to the idea of sustainable development due to a
Sustainable development more holistic view, as it strives for a compromise that avoids an unconditional increase in greenness at
the expense of functionality.
© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction 12 principles of GAC:

The concept of Green Analytical Chemistry (GAC) has been
established in the literature for over 20 years. The fundamental
principles of GAC have been well discussed in articles published in
1999 by Anastas [1], 2006 by Koel and Kaljurand [2], and 2008 by
Armenta et al. [3], presenting and further building on the idea of
implementing green chemistry in analytical procedures. The most
important assumptions of GAC were formulated in 2013 by
Gałuszka et al. [4] in the form of 12 main principles that express a
willingness to care for the environment and human safety during
the development and application of analytical procedures. In this
regard, proper implementation of GAC principles would include
practices such as minimizing reagent consumption, preferential use
of low-toxic and biodegradable reagents, saving energy, reducing
waste production, eliminating factors that may potentially cause
harm to the operator/analyst, and an increasing degree of inte-
gration, automation, miniaturization, and portability of analytical
tools and procedures. These 12 principles, as described by Gałuszka
et al. are listed below.
1. Direct analytical techniques should be applied to avoid
sample treatment.
2. Minimal sample size and minimal number of samples are
goals.
3. In situ measurements should be performed.
4. Integration of analytical processes and operations saves en-
ergy and reduces the use of reagents.
5. Automated and miniaturized methods should be selected.
6. Derivatization should be avoided.
7. Generation of a large volume of analytical waste should be
avoided and proper management of analytical waste should
be provided.
8. Multi-analyte or multi-parameter methods are preferred
versus methods using one analyte at a time.
9. The use of energy should be minimized.
10. Reagents obtained from renewable source should be
preferred.
11. Toxic reagents should be eliminated or replaced.
12. The safety of the operator should be increased.

* Corresponding author. Fax: þ48 12 686 27 50. In tandem with the development of greener methods, a number
E-mail addresses: wietecha@chemia.uj.edu.pl, renata.wietecha-posluszny@uj. of metric tools have been developed over the past years to measure
edu.pl (R. Wietecha-Posłuszny).

https://doi.org/10.1016/j.trac.2021.116223
0165-9936/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
P.M. Nowak, R. Wietecha-Posłuszny and J. Pawliszyn
Abbreviations
AC Analytical Chemistry
AGREE Analytical GREEness Calculator
GAC Green Analytical Chemistry
GAPI Green Analytical Procedure Index
GMO Genetically Modified Organism
HPLC High Performance Liquid Chromatography
LLE Liquid-Liquid Extraction
MAE Microwave-Assisted Extraction
MEPS Micro-Extraction on Packed Sorbents
MS Mass Spectrometry
NEMI National Environmental Methods Index
TCAD Tricyclic Antidepressants
SD Sustainable Development
SPE Solid-Phase Extraction
SPME Solid-Phase Microextraction
WAC White Analytical Chemistry
WCID Whole Column Imaging Detection
the degree of compliance of methods with the GAC concept in
qualitative and quantitative manners [5e11]. Of note, the (i) Eco-
Scale, the (ii) National Environmental Methods Index (NEMI), the
(iii) Green Analytical Procedure Index (GAPI), and the (iv) Analytical
Greenness Calculator (AGREE) have been adopted as potential tools
for greenness measurements. The (i) Eco-Scale is point scale
grading system in which an ideal green method is given an initial
value of 100 points; in this measurement, any deviation from GAC
ideals results in the imposition of penalty points and a decrease in
this value [10]. The (ii) National Environmental Methods Index
(NEMI) [8] and (iii) Green Analytical Procedure Index (GAPI) [9], in
turn, symbolically express the greenness of individual aspects of
the analytical procedure via the use of colored pictograms on a red
(bad) - green (good) scale. Lastly, the (iv) Analytical Greenness
Calculator (AGREE) [11] uses a simple algorithm that expresses the
overall greenness of the method in a quantitative manner on a 0e1
scale, while also utilizing a pictogram that directly refers to the 12
GAC rules.
An important issue related to the implementation of GAC
principles is the need to properly balance and reconcile the
greenness of the method with its potential usefulness/functionality,
which sometimes contradict each other. The usefulness of the
method is conditioned by the analytical efficiency expressed by
validation criteria (accuracy, precision, sensitivity), as well as
purely practical and economic considerations, e.g. cost of analysis,
speed of analysis, and general simplicity of the method. In this
regard, there are many different views and positions on how to
balance the greenness of the method with respect to other pa-
rameters. For example, the staunchest supporters of GAC may
promote the principle of selecting methods mainly in terms of
greenness, while those less convinced of the idea of GAC may be
guided by greenness only when the remaining parameters of the
methods under consideration are substantially satisfactory. In
addition to that, switching to greener methods also requires that
operators be trained in their development, optimization, and vali-
dation, which may be an obstacle for some. Furthermore, the
availability of green approaches may depend on economic, social
and geographical conditions.
In his work entitled “Do we need Green Analytical Chemistry?”,
Koel [12] draws attention to the need to develop functional
methods tailored to specific purposes that are at the same time as
economical as possible in relation to the amount of unnecessary
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Trends in Analytical Chemistry 138 (2021) 116223
data, material, reagents, waste, energy, costs, and time of analysis.
In his opinion, GAC should not be opposed to the “non-green”
traditional Analytical Chemistry (AC) approach, but directly result
from it as a logical consequence of striving to develop “fit-for-
purpose” methods. Accordingly, Koel asserts that defining GAC as a
separate branch of AC is unnecessary.
It should also be borne in mind that the concept of Sustainable
Development (SD) currently promoted worldwide is in fact multi-
dimensional [13]; by definition, it reinforces the need to strive for a
balance between the pursuit of prosperity, which goes hand in
hand with the progress of civilization, and care for the natural
environment. Anastas proposed recently an interesting approach to
this problem by proposing “the periodic table of the elements of
green and sustainable chemistry” [14]. As he claims, green chem-
istry will not be implemented at scale without the other elements
of sustainable chemistry. All efforts in chemistry need to take place
within an ethical, humanitarian, and moral framework. Just like the
countless possible substances of the known universe are comprised
of the known elements of the Periodic Table, there are countless
possible paths to a sustainable future when employing the ele-
ments of sustainable chemistry. Marcinkowska et al. [15], in turn,
call direct attention to the inconsistencies found in the ideals
proposed by GAC and SD, pointing out that SD should integrate the
environment, society, and economy as three main pillars. In this
work, the authors postulate that developed methods should be
green and “equitable” at the same time, i.e. easily available in terms
of price and applicability by everyday users. Some of the examples
given include methods based on smartphones, scanners, 3D
printers, and espresso machines. However, such easily available
solutions are not always possible, and as the authors point out [15],
the quality of results delivered, including for instance their sensi-
tivity and repeatability, will not always be sufficient for the planned
applications. This is obviously a natural consequence of trying to
find a compromise between contradictory ideas.
A reliable assessment tool to measure the degree of sustain-
ability of a given method is therefore difficult to develop and re-
quires that many factors be considered, such as whether the
method meets social demands through the required quality of re-
sults (validation criteria) and availability, whether it is economical,
and whether it is safe and environmentally friendly. It should be
emphasized that the greenness assessment tools discussed earlier,
namely the Eco-Scale, NEMI, GAPI and AGREE, do not capture the
potential of methods in a comprehensive manner, so they do not
allow methods to be assessed in accordance with the criterion of
SD. It is worth noting, however, that methods can be first pre-
selected in terms of other parameters (e.g. analytical and prac-
tical), and then, after narrowing down the pool to analytically and
practically acceptable methods, the greenness of such methods can
be compared and contrasted. Nevertheless, this approach raises
many questions and uncertainties as how to pre-select available
technologies in a fair and transparent manner that maintains the
desired balance of their features.
An alternative approach is to select methods using algorithms
that allow for a global assessment that takes into account all
desired criteria, including those expressing compliance with GAC
principles, and consider as optimal methods that achieve the
highest ranking in terms of this overall assessment. An overview of
available algorithms for global evaluations of analytical methods is
provided in a recently published article [16]. One such tool is the
recently introduced Red-Green-Blue (RGB) model [17], referring to
the color model commonly used in electronics, which extends the
concept of green chemistry to other primary colors (Red and Blue).
Red is assigned to analytical efficiency as expressed by validation
criteria (accuracy, precision, LOD, and others), green stands for
compliance with GAC principles, while blue represents productivity
P.M. Nowak, R. Wietecha-Posłuszny and J. Pawliszyn
and practical/economic efficiency. Following a computational
assessment, the method is assigned a color resulting from the share
of individual primary colors. An ideal, fully complete and consistent
analytical method is white, characterized by a high saturation of
each primary color. In the assumption that the resultant color is
further away from white in the RGB color space (darker), the overall
averaged potential of the method is lower. Along with the RGB
model, the authors also proposed the use of an algorithm using
Excel sheets that enables the method to be evaluated in a trans-
parent tabular manner. The algorithm is flexible in terms of the
selection of criteria to be assessed, their relative weights, and the
weights of the primary colors. However, this flexibility and the need
for the evaluator to select many variables may, in some cases,
hinder the application of the model.
The aim of current work is to present a new perspective for
implementation of SD principles in analytical chemistry, inspired
by the already known RGB model [17]. This idea is based on the
formulation of the so-called 12 principles of White Analytical
Chemistry (WAC), in analogy to the known 12 principles of GAC [4].
Such principles would allow one to express all main expectations
concerning desired method characteristics in a formal and sys-
tematic way, including green and other remaining criteria. These
pillars are divided into three complementary areas: red (analytical
efficiency), green (environmental friendliness and safety), and blue
(practical and economic aspects). Just as the color white is created
by mixing red, green, and blue light, the analytical method becomes
white, and thus complete, when it achieves each primary color.
WAC incorporates the main assumptions of GAC while also
addressing additional expectations, thus striving to maintain the
integrity of various parameters without directly prioritizing any of
the assessed attributes. Striving for SD is therefore striving for a
“white” method; hence we propose the use of the term “white” as a
synonym for a well-balanced analytical methodology fitted for
given application.
At the same time, we propose a new version of the RGB algo-
rithm (named RGB 12 in relation to the number of proposed rules)
that is fully integrated with the assumptions of WAC. The RGB 12 is
also based on a freely available Excel spreadsheet (available in
Electronic Supplementary Material) but has been simplified
considerably in comparison to the previous version [17]. This new
algorithm enables an easy and quick evaluation of methods against
each of the proposed 12 WAC principles, and from this perspective
assess the whiteness of the method, i.e. degree of sustainability.
In the next part of this paper, we apply RGB 12 to assess and
compare a model set of recently developed bioanalytical methods
aimed at determination of psychotropic substances e tricyclic an-
tidepressants (TCAD) in human biological fluids and tissues
[18e22]. These methods are based on different sample preparation
methods: Solid-Phase Extraction - SPE, Liquid-Liquid Extraction -
LLE, Microwave-Assisted Extraction - MAE, Micro-Extraction on
Packed Sorbents - MEPS, Direct Immersion Solid-Phase Micro-
extraction (DI-SPME), and use two different detection systems
(DAD and MS) but the same separation technique (HPLC). There-
fore, their characteristics mainly differ with respect to the sample
preparation step, which plays a critical role in the overall perfor-
mance of the analysis. Sample preparation determines the quality
and reliability of the obtained results of the chemical analysis,
especially in cases involving determination in complex biological
matrices, such as human body fluids or tissues. Moreover, studied
materials such as serum, saliva, or bone marrow are much more
complicated to analyze as compared to many other biological
samples since the former contain numerous proteins, salts, acids,
and bases, as well as various xenobiotics with properties similar to
the analytes of interest [23,24].
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Trends in Analytical Chemistry 138 (2021) 116223
The results of the WAC evaluation are further compared to re-
sults obtained using the AGREE assessment tool, which as previ-
ously mentioned attempts to provide measurements solely
according to the principles of GAC [11]. This allows us to contrast
both alternative concepts to each other and insightfully discuss
their usability and impact.
2. Proposition of the 12 WAC principles
To formulate the 12 WAC principles, we propose to synthesize
the 12 known GAC principles into 4 overarching “green” rules
covering the most important and mutually independent aspects of
GAC (G1-G4). These 4 principles are then joined by the 4 “red”
principles (R1-R4) and 4 “blue” principles (B1eB4) relating to
analytical efficiency and practical/economic criteria, respectively, to
compose the 12 WAC principles in total. It is worth emphasizing at
the beginning that, according to the WAC concept, all colors and
principles are equally important, to maintain the idea of sustain-
ability. However, the actual usefulness and functionality of a
method may be determined only by certain principles that are
crucial in a given specific case and related circumstances, e.g. by
LOD and cost-efficiency. Therefore, each principle should be
considered during the method evaluation, but the most important
ones for a specific application should be assessed more severely,
adequately to the requirements. The overall compliance of the
method with the proposed rules is expressed by a quantitative
parameter called “whiteness”, which is the simplified measure of
how the method fits the planned application.
2.1. Red
R1. Scope of application. Analytical methods should have the
widest possible range of applicability expressed in the number of
simultaneously determined analytes, range of linearity of the de-
terminations, compatibility with various types of samples and
resistance to the presence of potential interferences.
R2. LOD and LOQ. Analytical methods should have the lowest
possible limits of detection and quantification (LOD and LOQ).
R3. Precision. Analytical methods should be characterized by the
best possible precision expressed in repeatability and reproduc-
ibility of the results.
R4. Accuracy. Analytical methods should be as accurate as
possible (minimal relative error of determinations and recovery as
close to 100% as possible).
2.2. Green
G1. Toxicity of reagents. Analytical methods should be charac-
terized by the lowest possible toxicity of reagents used and the
maximum share of biodegradable/renewable reagents and
materials.
G2. Number and amount of reagents and waste. Analytical
methods should be characterized by the lowest possible con-
sumption of reagents and production of waste (regardless of how
toxic they are).
G3. Energy and other media. Analytical methods should be
characterized by the lowest possible consumption of electricity and
other utilities. On-site, automated and high-throughput methods
are preferred for saving energy.
G4. Direct impacts. The use of analytical methods should not
directly affect humans, animals, and genetic naturalness. Exposure
of humans (personnel) to harmful factors and the use of animals
and/or genetic modifications should be avoided.
P.M. Nowak, R. Wietecha-Posłuszny and J. Pawliszyn
2.3. Blue
B1. Cost-efficiency. Analytical methods should be as cost-efficient
as possible (where the total cost of analysis should take into ac-
count instruments, materials, media, and personnel).
B2. Time-efficiency. Analytical methods should be characterized
by the highest possible time-efficiency (the smallest total time of
analysis, including method development and all stages of the
analytical workflow).
B3. Requirements. Analytical methods should be characterized by
the minimal practical requirements including the amount of sam-
ple used, access to advanced equipment, personnel qualifications,
and laboratory infrastructure.
B4. Operational simplicity. Analytical methods should be char-
acterized by the highest possible level of miniaturization, integra-
tion, automation (on-line methods, AI technologies), and
portability (on-site measurements).
As can be surmised, we propose to formulate the issues covered
so far by the GAC principles relating directly to practical benefits,
such as integration, automation, miniaturization and portability of
the method, in the form of a blue principle (B4), which seems
reasonable taking into account their massive impact on practical
usability. At the same time, it is worth emphasizing that their
translation into ecological issues comes down to the implementa-
tion of other green principles, such as minimizing toxicity (G1),
waste (G2) and saving energy (G3). Therefore, one should also
consider these issues when analyzing individual green principles,
e.g. the positive impact of on-site and high-throughput analysis on
reducing energy intake, etc.
It is also worth emphasizing that when assessing the G2 rule, it
is important to reliably estimate the actual amounts of reagents and
waste, because it is this rule that can largely determine the position
of a given method in the ranking, especially when comparing
similar methods differing in the stage of sample preparation. For
example, the current development of the liquid phase micro-
extraction technique goes hand in hand with a significant minia-
turization of solvents volume [25], and hence, allows to maintain
the effectiveness of this technique with a significant increase in its
greenness compared to the classic version.
Moreover, we propose to draw attention to two contemporary
issues which, in our opinion, affect the green nature of the method
as well. These encompass the use of laboratory animals, e.g. rabbits
for the production of antibodies used in enzyme immunoassay
methods, as well as genetically modified organisms (GMOs), e.g. for
the production of recombinant proteins. Although the benefits of
using animals and GMOs are enormous, it is a departure from the
idea of caring for the living environment and striving to maintain
its natural form undisturbed by humans.
Other key issues that must be addressed pertain to how the
implementation of given method might be limited by the famil-
iarity of the personnel with the techniques used in the method, and
the length of method development. For instance, SPME applications
necessitate the construction of equilibration time profiles for ana-
lytes of interest, optimization of salt concentration, investigations
of pH effect, etc., as part of the optimization process. It might not be
a good investment of time to develop such method if only a few
determinations are needed, but it is justified if a fully automated
method is developed to process large numbers of samples or used
regularly. Owing to this, we advise that the length of method
development be considered when assessing time-efficiency (B2),
and likewise, that personnel qualification be considered when
assessing practical requirements (B3). In addition, the assumption
of “equitable” AC [15] were included in blue principles; accordingly,
the availability of equipment and infrastructure was included in the
B3 principle, their cost in B1, and portability in B4.
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Trends in Analytical Chemistry 138 (2021) 116223
Finally, in order for the proposed principles to be comprehen-
sive, we have integrated some related issues in the form of single
principles, e.g. the number of analytes determined, range of line-
arity and possibility of analysis in various matrices, as a common
principle R1 that strives for a wide applicability range.
3. The RGB 12 algorithm e working principle
In order to evaluate the method using the RGB 12 algorithm, the
red, green and blue tables prepared in the Excel template spread-
sheet must be filled out (see Fig. 1). The template allows for
simultaneous evaluation and comparison of up to 10 methods. The
only necessary step is to enter the scores in the appropriate gray
columns (Fig. 1), 0 being the worst result, and 100 meaning that the
method is well fitted for a planned application in respect to
particular principle. Awarding the additional merit points (scores
above 100) is also possible in justified cases (see the next para-
graph). The criteria related to the use of animals and GMOs are an
exception; in this case, if they are used at any stage of the pro-
cedure, enter 1, and if not, then 0.
These values should be assigned in the most objective and
reasonable manner. It is extremely important that the individual
criteria are always assessed in relation to the intended application
of the method. We suggest to always answer the question whether
the given values are appropriate for a given type of analysis,
chemical nature of the analytes, nature of the sample matrix, etc. As
already Koel proposed in his interesting discussion [12], the key
question to be answered should be whether the method is fit for a
particular purpose. On that account, we do not recommend the
minimum values of given parameters deserving a given score, this
decision remains for the evaluator and should be adapted to the
specific case and context of the assessment. For example, a score of
50 may be awarded in a situation where a given result seems to be
unsatisfactory and tolerable only in some cases, a score of 75 could
be assigned if a result seems quite good but still not sufficient for all
expected situations, while a score of 100 should be assigned, as
mentioned above, to a method that seems to be appropriate for its
target application and exhibit the fully satisfactory compliance with
given principle. Noticeably, the scores above 100 can be awarded
for the method which exceeds expectations, e.g. offers LOD much
better than required, if there is anticipation that the application
could benefit from such additional capability or it is expected it may
need such capability in the future. The assigned scores should not
be linearly correlated with the values of the assessed parameters,
but reflect the actual benefits.
To illustrate this issue, one may mention a Whole Column Im-
aging Detection (WCID) technology [26], which in combination with
capillary isoelectric focusing is now used as a “gold standard” in
biotech industry for product development and quality control
[27,28]. Compared to the MS detector, this detection technique is
much less sensitive and selective, which could significantly reduce
the redness of the entire method in direct comparison. Nevertheless,
for quality control applications, WCID is used because sensitivity is
appropriate as the samples are low complexity high concentration
protein mixtures. The main added value is the significant improve-
ment in the “green” and “blue” aspects, which makes the method as
a whole so attractive. In assessing the LOD and LOQ values, it will
therefore be fair to give a score of 100 for WCID because they are fully
sufficient regarding the current requirements, while e.g. 120 for MS
detector. These 20 extra merit points note potential benefits that
may appear in the future or when analyzing “unusual” samples. Such
a common-sense approach to the assessment of individual criteria
will ensure that the assessment of the overall potential reflected by
whiteness, will not be underestimated or overestimated, and thus
will be more objective.
P.M. Nowak, R. Wietecha-Posłuszny and J. Pawliszyn
Fig. 1. Evaluation tables for methods according to the red, green, and blue principles using the RGB 12 algorithm. In order to carry out the assessment, appropriate scores must be given in
the gray columns. In the above example, three model methods were assessed by assigning them exemplary scores: the worst possible (1), appropriate (2), and intermediate (3).
When scoring according to the above guidelines seems difficult,
e.g. for green criteria, one can consider the predicted number of
alternative methods available that offer better results. For instance,
if the method under consideration is in the top 20% in a given
respect, a score of 100 or more may be given.
To facilitate fair awarding of scores in the case of rules strictly
related to the value of certain parameters, e.g. LOD, it is recom-
mended that these values be entered in the white columns to the
left of the columns with scores, this is however not mandatory for
performing assessment (see Electronic Supplementary Material for
an illustration). The outcome of compliance with rules that include
several features of the method, for example B4, (operational
simplicity), is calculated as the average of the awarded scores.
After entering the appropriate scores, the evaluation results are
calculated and formatted automatically in the form of tables, the
interpretation of which is quite simple (Fig. 2). The degree of
compliance of the method with a given WAC rule is presented
numerically as well as visually via the saturation of a given primary
color (a value of 0 corresponds to black, 100 and more to full satu-
ration). As seen below, the values of the arithmetic means for the
given primary attributes e R(%), G(%), B(%), are presented individu-
ally, and at the bottom, an overall score as the average result of the
assessment of all 12 principles - whiteness(%), is also given.
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Trends in Analytical Chemistry 138 (2021) 116223
It is worth noting that the color assigned by the RGB 12 algo-
rithm is only an additional way of presenting the results. The
saturation used for background formatting is linearly dependent on
the numerical values, according to the RGB color coding model.
Thus, it allows for faster interpretation and easier recollection of
results. This feature is a departure from the first version of the RGB
algorithm [17], which differentiated nine resultant colors as distinct
outcomes of the evaluation. The RGB 12 algorithm is simplified in
this respect; the overall evaluation is expressed only by the
“whiteness” parameter, formatted for simplification in black and
white. Therefore, the analysis is easier for color-blind people or
when printing in black and white. However, the contribution of the
individual primary attributes represented by red, green, and blue
colors is still clearly represented by the values of R(%), G(%) and B(%)
and the formatting of the corresponding cells.
4. Exemplary analysis of experimental methods with the RGB
12 algorithm
The evaluation results of the seven selected model methods are
presented in Fig. 3 (presentation of the results for each method)
and Fig. 4 (comparison of the main evaluation parameters of all
methods) [18e22]. The input experimental data used for
P.M. Nowak, R. Wietecha-Posłuszny and J. Pawliszyn
Fig. 2. Visualization of the evaluation results of the three model methods (see Fig. 1) according to the RGB 12 algorithm (detailed description in the text).
performing evaluations are shown in the Electronic Supplementary
Material (Figs. S1eS3). The presented data shows that overall as-
sessments, expressed by the whiteness parameter, yielded fairly
similar results, ranging from 82.2% (LLE-HPLC-DAD) to 96.3%
(SPME-HPLC-MS), indicating that the analyzed methods have
similar overall potential. However, some noteworthy dissimilarities
become visible when the contribution of each primary color is
closely assessed.
The lowest R(%) result was obtained for the LLE-HPLC-DAD
method - 77.5%, which is related to the low-rated accuracy (prin-
ciple R4), assessed at the score of 40. Despite the highly rated other
analytical criteria, high inaccuracy can significantly affect the
functionality of the method, hence, it may exclude certain uses. A
similarly big shortcoming of the LLE-HPLC-DAD method with re-
gard to the green color is the amount of reagents and waste
(principle G2), which is a common drawback of the standard LLE
method in a large-scale format. The blue aspects were assessed
better, in particular, very good cost- and time-efficiency, scored at
100, is the best among all the methods.
On the other extreme, there is the SPME-HPLC-MS method,
assessed overall best, with the result of R(%) estimated at 107.5%,
and G(%) and B(%) above 90. Such a good balance of the three pri-
mary attributes allowed to achieve a high value of whiteness, close
to 100%, which means that the method is almost perfectly suited to
Fig. 3. Comparison of the seven model methods for determination of TCAD in body fluids according to the 12 principles of WAC, performed using the RGB 12 algorithm.
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Trends in Analytical Chemistry 138 (2021) 116223
the planned applications. The least rated principles are G3 (energy
consumption) and B1 (cost-efficiency), which results from the use
of the MS detector, expensive and energy consuming, as well as
quite expensive fibers used in the SPME technique. On the other
hand, the high sensitivity and the ability to analyze much more
chemically complex samples offered by the MS detector were re-
flected in the high scores for other principles, R1 (scope of appli-
cation) and R2 (LOD & LOQ), above 100, as it can be additional
utility in the future and potentially increase the quality of the
method. This very good overall assessment is also related to the
nature of SPME as an extremely versatile sample preparation
technique, that is economical with regard to the use of reagents, as
well as safe and fast [29,30]. As the SPME technology matures, the
price consideration could become advantage considering the low
cost of production. Particularly with implementation of the tech-
nique in high throughput applications, this could become attractive
feature as the devices can be used multiple times, when applied in
automated fashion with optimized method.
The MEPS-HPLC-MS and MAE-HPLC-MS methods also obtained
the high R(%) values exceeding 100, as before, it is associated with
the use of efficient extraction techniques (MEPS and MAE) guaran-
teeing, similar to SPME, high accuracy and precision, and also with
the use of the MS detector. The MEPS-HPLC-MS method turned out
to be slightly inferior to the SPME-HPLC-MS overall (90.7% vs. 96.3%)
P.M. Nowak, R. Wietecha-Posłuszny and J. Pawliszyn
Fig. 4. Comparison of the main assessment outcomes obtained from the RGB 12 analysis, the white line indicates 100% - a full appropriateness for planned application. The values
above 100 indicate additional capabilities exceeding current requirements.
due to the lower assessed B1 and B2 rules corresponding to lower
cost- and time-efficiency. The whiteness of the MAE-HPLC-MS
method turned out to be yet lower (84.8%), mainly due to the
much worse greenness, and in particular, much larger amounts of
solvents used (principle G2), but still smaller than in the LLE tech-
nique. It is also worth emphasizing that the MEPS-HPLC-MS method,
although overall weaker than SPME-HPLC-MS, shows the higher
G(%) value due to the minimal use of solvents (G2 scored at 120).
The analogous method using the DAD detector (MEPS-HPLC-
DAD) was the only one that achieved the value of 100% in the green
attribute, thus it is the most ecological, mainly because its energy
consumption is lower thanks to the use of a different detector. This,
combined with the better assessed blue criteria (it is cheaper and
faster), made it possible to fully compensate for some deficiencies
in the red aspects and to obtain a slightly better overall result than
the MEPS-HPLC-MS method (91.1% vs. 90.7%). Similar relationships
between the colors, i.e. the worse red criteria but better green and
blue ones, are observed in the comparison of the MAE-HPLC-DAD
and MAE-HPLC-MS methods. Here, however, the inferior accuracy
(principle R4) obtained with the MAE-HPLC-DAD method is not
necessarily due to a different detector but to other factors.
To sum up, the SPME-HPLC-MS method turned out to be the
best overall, i.e. the most white. Nevertheless, the general power of
the other methods is similar and each of them deserves consider-
ation in the context of a possible practical application. However,
apart from the whiteness value, it is worth considering the
assessment of all individual rules. This is facilitated by the use of
appropriate formatting, where the color of the respective cells is
the darker the lower score is awarded. This is shown in Fig. 3, e.g. in
the case of LLE-HPLC-DAD method, where it is easy to note that the
principles R4 and G2 can be the bottleneck of the whole method
(scored only at 40). Therefore, the proposed method of presenting
the results in the form of colored tables (Figs. 2 and 3) seems
convenient to use and quickly interpret.
Assuming that “Method” is defined as a combination of a
number of “Techniques”, e.g. SPME-HPLC-MS is a combination of
the three techniques: SPME, HPLC, and MS, one should emphasize
that the evaluation of the overall method might be limited by one of
the techniques used. For example, HPLC-MS is a large, sophisti-
cated, expensive and energy-consuming instrument. On the other
7
Trends in Analytical Chemistry 138 (2021) 116223
hand, given that SPME extraction amounts are typically low, a high
sensitivity determination (readout) technique such as like HPLC-
MS is required to make it practical for some applications, which
might result in a less attractive method. One possible solution
might be to combine SPME with a spectroscopic or electrochemical
readout technique, although this alternative would result in more
specific determinations and reduce the universality of the
approach. Therefore, it is advisable that investigators clearly specify
whether the whole method or only a specific technique is under
assessment; in cases where full methods are considered, careful
interpretation of the assessment results should be carried out. With
reference to the above example, the rankings attained by the model
methods are not indicative of the superiority or inferiority of the
sample preparation techniques used, but only of the effectiveness
of the technique when used together with the specific combination
of instrumental techniques described in this work.
The above analysis shows that the RGB 12 algorithm allows one
to easily and quickly evaluate and compare methods in terms of
compliance with specific WAC rules, their overall potential for a
given primary color, and the global averaged potential, expressed
by whiteness. At the same time, the results of the assessment allow
investigators to identify the strengths and weaknesses of the given
methods, predict their predispositions, and choose the appropriate
ones in terms of the planned applications and related specific
requirements.
As shown in this model analysis, the proposed WAC tool and
related RGB 12 algorithm are ideally suited to evaluate specific
procedures and methods on the basis of experimental data. As such,
it should be highlighted that experimental outcomes may differ
from theoretical predictions based on literature data, thus resulting
in different classifications. The position of a method in the ranking
may, for instance, depend on the specifics of the sample material.
However, this does not exclude the possibility of trying to evaluate
a hypothetical method only theoretically, e.g. to contrast theory and
practice, or consider potential benefits offered by a particular
methodology before its development. An accurate evaluation of a
given experimental method is only possible when said method is
optimized for a given task, whereas a theoretical evaluation can be
accomplished at any moment and context as a starting point of
investigation.
P.M. Nowak, R. Wietecha-Posłuszny and J. Pawliszyn
Fig. 5. Comparison of the seven model methods for determination of TCAD in body fluids according to the 12 principles of GAC, performed using the AGREE algorithm.
5. Comparative analysis of experimental methods with the
AGREE algorithm
As mentioned before, AGREE is used to evaluate the greenness of
a given method according to the 12 GAC principles [11]. The concept
of selecting an optimal method by AGREE is therefore primarily
based on choosing the greenest method, assuming an earlier pre-
selection of methods in terms of analytical and practical aspects.
In this case, all model methods were assumed to have passed such
pre-selection, namely the standard validation procedure. At the
same time, it is worth noting that some clear differences between
methods relevant to the WAC concept, in particular pertaining to
the red and blue criteria, were ignored. Therefore, in order to
directly compare the results of assessments with RGB 12 and
AGREE, only the G(%) index corresponding to greenness should be
used. It should also be highlighted that assessments of individual
GAC principles with the use of AGREE are made based on the
models proposed by the authors, which determine in advance, for
example, what amount of waste corresponds to a given assessment
score, etc. In the case of RGB 12, it is the user who determines the
final score of assessment of a given principle, assuming that this
process should be as objective and critical as possible for the given
specific context and resulting circumstances.
A comparison of the results obtained with AGREE is shown in
Fig. 5. The obtained values do not differ significantly, reaching a
maximum point range difference of 0.12 on a scale of 0.00e1.00
between the best and worst methods. For comparison, the differ-
ences in G(%) values in the RGB 12 model were over twice as large
(72.5% vs. 100%). Importantly, both AGREE and RGB 12 indicate the
MEPS-HPLC-DAD method as the greenest from the whole group,
while the MEPS-HPLC-MS and SPME-HPLC-MS methods as
respectively less green. The other methods show very similar
greenness according to AGREE, between 0.46 and 0.51, among
them, MAE-HPLC-DAD incurred the worst ranking. Here, the two
algorithms diverged in terms of their ranking: RGB 12 indicated the
MAE-HPLC-MS method as the worst (G ¼ 72.5%), while MAE-HPLC-
8
Trends in Analytical Chemistry 138 (2021) 116223
DAD turned out clearly better than the analogous method with a
different detector (G ¼ 81.3%). It can also be concluded that the
results obtained with AGREE are generally poorer than RGB 12,
with the best result according to the former tool being 0.60/1.00,
and the latter 100%, respectively. From the AGREE position, the best
method is therefore still far from the assumed ideal, while from the
perspective of the WAC concept, it is fully satisfactory in the context
of the current possibilities and available alternatives. Therefore, it
can be assumed that AGREE carries out the evaluation process more
idealistically, while RGB 12 more pragmatically, in relation to the
actual possibilities offered by the current reality. Furthermore, RGB
12 allows for awarding scores above 100 for significant improve-
ments in relation to available options, hence it supports efforts to
make a constant methodological progress.
Moreover, while in the case of RGB 12 each of the four green
principles influenced the final results of the method evaluation, in
the case of AGREE, only a few of the twelve principles were
differentiating and relevant for the evaluation result, such as
sample amount, waste volume, toxicity, and consumption of energy
and time (see Fig. 5). It is also worth noting that some of these
parameters were assessed in the case of RGB 12 as blue criteria
(consumption of sample and time), due to their direct translation
into practicality and correlation with other green factors.
6. Discussion (WAC vs. GAC)
Considering only the evaluation of greenness, the results ob-
tained using RGB 12 and AGREE, related to the WAC and GAC ideas,
respectively, should be considered as generally consistent. Both
algorithms indicated the same methods as greenest. However, one
should be aware of the differences in criteria and evaluation pro-
cedure which may potentially cause larger inconsistencies between
both metrics in other comparisons. The AGREE method takes more
parameters into account, relying on pre-defined inflexible models
to evaluate compliance with the particular principles. RGB 12 is
based on a more synthetic and flexible approach. It considers only
P.M. Nowak, R. Wietecha-Posłuszny and J. Pawliszyn
four green parameters independent of each other (toxicity, amount
of reagents/waste, energy consumption, direct impacts/risks),
bearing in mind, however, that they are influenced by many factors.
For example, the use of derivatizing reagents is defined by a
separate rule in the case of GAC, while in the concept of WAC it
should be included as one of the elements influencing the toxicity
of the reagents used. Likewise, automation, instrument portability,
integration of analysis steps and throughput, although not assessed
separately in RGB 12, should have an impact on assessments of
energy consumption and the number of reagents consumed, and
waste generated. In addition, WAC introduces two new factors, the
use of animals and GMOs, which are also associated with the idea of
greenness. However, in the case of the current comparison, they
were not applicable to differentiate the methods.
As such, the GAC concept seems most effective and preferred
when greenness is supposed to be a decisive factor, assuming that
any differences in analytical and practical criteria are insignificant at
the stage of evaluation. Alternatively, it can be assumed that
methods are initially graded for greenness, and only those that have
received a certain minimal score are further assessed with a different
algorithm against other parameters determining the final ranking.
The idea of WAC assumes that striving for SD must include a
compromise between greenness and usefulness, expressed quan-
titatively by the level of a method's whiteness. A key issue is a good
balance of the red, green, and blue attributes. Whiteness is an easy-
to-use and comparative indicator of how good the method under
consideration is, but relying only on it when selecting the optimal
method is associated with certain risks. It may happen that the
whiteness of the method will be high, but the application of the
method may be difficult or even impossible due to the bottleneck
which may be one or more criteria, e.g. accuracy, LOD, cost-
effectiveness, etc. Therefore, always one should take into account
the assessment of all rules and the possibility of the occurrence of
such traps.
Although the RGB 12 algorithm does not allow for the direct
ascribing of various weights to the given rules, as does AGREE [11],
the specificity of a given method should be reflected in the level of
criticality when assessing the key rules for its application. For
example, when the accuracy of the method is the most important
thing, small changes in the quantification error should strongly
affect the scores, while the evaluation of less important principles
should not deviate significantly from 100. It is also important to
consider the green criteria with the well-balanced significance, as
dictated by researcher's ethical responsibility.
A potential problem may be the lack of sufficient self-criticism of
the authors in the assessment of their own methods, manifested in
the overstatement of scores assigned to particular principles.
Indeed, the proposed algorithm presents the evaluator's point of
view in a specific situation and context. Nevertheless, the same issue
is often encountered in publications when authors subjectively
discuss the potential of newly developed methods, sometimes with
too much enthusiasm. The use of the RGB 12 algorithm, noticeably,
forces the evaluator to assess criteria that might otherwise be left
out of the discussion or neglected; hence, the assessment is sub-
jective but always comprehensive. Further, the assessment outcome
is not ultimate, as methods can be easily reassessed in different
contexts, in a more or less critical way; for instance, methods can be
reassessed by the authors of competing methods for the same
application, etc. Therefore, we recommend that the proposed
application for which a method is being assessed be clearly desig-
nated. In addition, we encourage to publish real experimental data
on the basis of which the evaluation of individual quantitative
criteria was made, not only the parameters normally determined
during validation, but also the estimated cost, time of analysis,
sample consumption, the amount of reagents and waste generated,
9
Trends in Analytical Chemistry 138 (2021) 116223
toxicity measured e.g. by the number of pictograms and the number
of additional environmental hazards. For this purpose, tables inte-
grated with the proposed RGB 12 algorithm can be used, presented
in Fig. 1. Such data can be, for instance, attached as a supplement to
publications. When it is difficult or even impossible to give exact
values for certain parameters, estimates and even rough approxi-
mations can be performed, but this should be noted.
Noticeably, it might be impossible and even irrational to pro-
pose at this moment a rigid framework for assessment of each of
the 12 principles, given the variety of methodologies and related
analytical problems that must be addressed. Nevertheless, the
development of such objective standards for the assessment of
individual rules for methods of a given type is an interesting
prospect that is worth developing in the future. Instead, we suggest
now the use of a flexible framework that fits the needs of the user's
laboratory and is adjusted to its specificity, because as it is worth
emphasizing once again, the method should be assessed in terms of
its intended use. Taking into account the above guideline will
ensure high objectivity of assessments, without losing the desired
flexibility for a variety of analytical goals.
In this context, the herein presented WAC concept presents an
advantage over GAC, as it considers all attributes equally, in the
same formalized and systematic way, allowing methods to be
assessed more comprehensively and flexibly. Such an approach is
closer to the idea of SD, and as we believe, can help to treat the
green chemistry aspects with due regard in the future.
7. Conclusions
The WAC concept proposed in this article, along with the RGB 12
algorithm as a dedicated method evaluation tool, is an extension
and complement to the GAC principles currently promoted in
analytical chemistry. In our opinion, WAC fits better with the
paradigm of SD, assuming that the basis for designing and devel-
oping new methods should always be a balance between greenness
and other essential attributes influencing the actual usefulness and
functionality of the method. Therefore, the analytical (red) and
practical (blue) criteria are considered in an analogous way to
greenness, based on formalized principles and the same evaluation
tool. According to the WAC concept, the method becomes white, i.e.
appropriate, if meets varied criteria, thus maintains the complete-
ness symbolized by the color white. We suggest simultaneously
that greenness should always be treated with great attention;
however, treating it with undue priority while incurring a signifi-
cant loss of functionality should be avoided, as should avoid dis-
regarding greenness at all. The described approach should assist
analytical chemistry community in choosing the optimal method
for a given application, evaluating newly developed methods,
comparing new and known methods, as well as to place new ad-
vancements in a perspective, and decide about the ways forward. It
should also aid industrial and government scientists in making
decision about method acceptance and further investigation of
analytical technologies, in view of the sustainable future.
8. Electronic Supplementary Material (ESM)
The Excel worksheet as a template for assessments using the
RGB 12 algorithm. The additional data used for assessing the model
methods.
Author contribution statement
Paweł Mateusz Nowak: Conceptualization, Data curation,
Investigation, Methodology, Software, Visualization, Writing -
original draft, Writing - review & editing.
P.M. Nowak, R. Wietecha-Posłuszny and J. Pawliszyn
Renata Wietecha-Posłuszny: Conceptualization, Data curation,
Investigation, Methodology, Software, Visualization, Writing -
original draft, Writing - review & editing.
Janusz Pawliszyn: Conceptualization, Formal analysis, Supervision.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
P.M. Nowak acknowledges the financial support from the Na-
tional Science Centre, Poland (OPUS, 2020e2024, grant no. 2019/
35/B/ST4/01022).
R. Wietecha-Posłuszny acknowledges the financial support from
the National Science Centre, Poland (Sonata Bis 6, grant no. 2016/
22/E/ST4/00054).
J. Pawliszyn acknowledges the financial support from Natural
Sciences and Engineering Research Council of Canada, Industrial
Research Chair Program (IRCPJ184412-15).
The publication was funded by the Priority Research Area
Anthropocene under the program “Excellence Initiative e Research
University” at the Jagiellonian University in Krakow”.
The authors also acknowledge Prof. P. Ko
scielniak and Prof. M.
Wo
zniakiewicz for fruitful discussions.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.trac.2021.116223.
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