energies

Review
Quantification Methodology of Ammonia Produced from
Electrocatalytic and Photocatalytic Nitrogen/Nitrate Reduction
Wahyu Prasetyo Utomo 1 , Hao Wu 2, * and Yun Hau Ng 1,3, *

1 School of Energy and Environment, City University of Hong Kong, Kowloon, Hong Kong 999077, China
2 Macao Institute of Materials Science and Engineering (MIMSE), Zhuhai MUST Science and Technology
Research Institute, Faculty of Innovation Engineering, Macau University of Science and Technology, Taipa,
Macau 999078, China
3 Shenzhen Research Institute, City University of Hong Kong, Shenzhen Hi-Tech Industrial Park,
Nanshan District, Shenzhen 518057, China
* Correspondence: [email protected] (H.W.); [email protected] (Y.H.N.)

Abstract: Nitrogen reduction reaction (NRR) and nitrate reduction reaction (NO3 − RR) provide a
potential sustainable route by which to produce ammonia, a next-generation energy carrier. Many
studies have been conducted over the years, mainly emphasizing material design and strategies
to improve catalytic performance. Despite significant achievements in material design and corre-
sponding fundamental knowledge, the produced ammonia is still very limited, which makes it prone
to bias. The presence of interferants (e.g., cations and sacrificial reagents), the pH of the solution,
and improper analytical procedure can lead to the over or underestimation of ammonia quantifica-
tion. Therefore, the selection of the appropriate ammonia quantification method, which meets the
sample solution condition, along with the proper analytical procedures, is of great importance. In
this review, the state-of-the-art ammonia quantification method is summarized, emphasizing the
advantages, limitations, and practicality for NRR and NO3 − RR studies. Fundamental knowledge of
the quantification method is introduced. Perspective on the considerations for selecting the suitable
quantification method and for performing the quantification process is also provided. Although
non exhaustive, this focused review can be useful as a guide to design the experimental setup and
procedure for more reliable ammonia quantification results.
Citation: Utomo, W.P.; Wu, H.; Ng,
Keywords: ammonia; quantification; detection; nitrogen reduction; nitrate reduction
Y.H. Quantification Methodology of
Ammonia Produced from
Electrocatalytic and Photocatalytic
Nitrogen/Nitrate Reduction. Energies
2023, 16, 27. https://doi.org/ 1. Introduction
10.3390/en16010027 Ammonia (NH3 ) is an essential feedstock chemical for the fertilizer, pharmaceutical,
Academic Editor: Qingran Zhang
and nitrogen-containing chemicals industries. Today, it is also considered a next-generation
energy carrier due to its high energy density for hydrogen (17.6%) [1–6]. Up to now,
Received: 7 November 2022 ammonia production has heavily relied on the conventional Haber-Bosch process, which
Revised: 7 December 2022 requires a high energy supply (temperature 400–500 ◦ C and pressure 100–200 atm) [7–9],
Accepted: 12 December 2022
and is responsible for 1.2% of global CO2 emissions [10–13]. Nitrogen reduction reactions
Published: 20 December 2022
(NRR) and nitrate reduction reactions (NO3 − RR) to ammonia have attracted increasing
attention because they can be performed under ambient conditions and are potentially
more sustainable [14–16]. NRR and NO3 − RR can be performed in photocatalytic (PC),
Copyright: © 2022 by the authors. electrocatalytic (EC), or photoelectrocatalytic (PEC) systems [17–24]. In the last five years,
Licensee MDPI, Basel, Switzerland. publications relating to this field have significantly increased, indicating the noticeable
This article is an open access article potential of these processes (Figure 1).
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Energies 2023, 16, 27. https://doi.org/10.3390/en16010027 https://www.mdpi.com/journal/energies

Energies 2023, 16, 27 2 of 22

Figure 1. Number of publications in (a) NRR and (b) NO3 − RR studies. Data were obtained from
the Web of Science based on an article title of “nitrogen reduction reaction” and “nitrate reduction
reaction”, respectively.

The advancement of the photo(electro)catalytic NRR and NO3 − RR has been reported
by many researchers over the years [25–28]. Most of the works have emphasized material
design and strategies to improve catalytic performances, i.e., ammonia production rate
and selectivity. For example, Utomo et al. reported that copper loading on the surface
of oxygen-deficient TiO2 could enhance its catalytic performance over EC-NRR due to
a strong metal support interaction (SMSI) between the copper nanoparticles and TiO2
substrates [28]. The SMSI led to an enlarged electrochemically active surface area, increased
electron density, and promoted electron transfer and nitrogen activation, which resulted
in an enhanced NH3 yield of 13.6 µg mgcat −1 h−1 (∼ 0.06 mg L−1 NH3 -N) at −0.5 V vs.
RHE compared to the bare TiO2 . In PC-NRR, Liu et al. reported the deposition of the
Bi4 O5 Br photocatalyst on the surface of hydrophobic zeolitic imidazolate framework-8
(ZIF-8), which created the triphasic contact between nitrogen gas, liquid (water-based)
electrolyte, and the solid photocatalyst [29]. The hydrophobic nature of ZIF-8 retarded the
diffusion of the liquid (water-based) electrolyte and facilitated the direct contact between
the nitrogen gas with the Bi4 O5 Br photocatalyst. As a result, ammonia generation reached
327.388 µmol gcat −1 h−1 (∼ 0.46 mg L−1 NH3 -N), which was 3.6-fold higher compared
to pristine Bi4 O5 Br. Recently, Cu-based materials have also been reported as promising
catalysts in EC-NO3 − RR. A Cu/Cu2 O catalyst showed a high ammonia yield of up to
0.2449 mmol·cm−2 h−1 (∼ 171.4 mg L−1 NH3 -N) at −0.85 V vs. RHE. The high perfor-
mance was attributed to the electron transfer from Cu2+ to Cu0 , which facilitated the
formation of NOH intermediate (*NOH) as one of the key intermediates and suppressed
the hydrogen evolution reaction [30].
Regardless of the significant achievements in material design, and crucially funda-
mental knowledge that has been established, production rates and selectivity of the current
catalytic system are still far from gaining industrial interest. For instance, the production
rate of NRR mostly ranges from a few to 35 µg mgcat −1 h−1 , which is approximately still at
the micromolar level [25]. In comparison, the NO3 − RR performs better, with production
rates reaching the millimolar level [31]. The wide range of ammonia production leads
to the need for compatible quantification methods. The low ammonia production rates
make it prone to interference from impurities in the catalytic system, which deviates the
measured ammonia from its true value [32]. Moreover, the ammonia quantification, which
is performed without carefully considering the condition of the sample solution and the
specification of the quantification method, may raise concerns about the reliability of
the measurements.
Several ammonia quantification methods have been used in many applications. Spec-
trophotometric methods, better known as the indophenol blue and Nessler’s method, are
two typical standard ammonia detection methods widely used in water quality control [33].
Today, these methods were also frequently used in NRR and NO3 − RR. The ion chromatog-
raphy (IC) and 1 H nuclear magnetic resonance (1 H NMR) methods have also been used
Energies 2023, 16, 27 3 of 22

as primary or supporting detection methods in NRR [25,31]. Other methods, such as ion
selective electrodes (ISE) and fluorometric methods, are also known for ammonia detec-
tion, but application is still limited in terms of water quality control [14]. The selection
of the most suitable ammonia quantification method depends on the catalytic system
under investigation and the specification of each method. Some papers have reported
the limitations of spectrophotometric and IC methods for NRR studies by subjecting the
methods to some possible interferants, such as pH, cations, and sacrificial reagents [34,35].
Some reviews have also introduced several ammonia detection methods as an additional
part of their discussion [14,25,31,36]. However, to the best of our knowledge, a focused
review concentrating on ammonia quantification methods in NRR and NO3 − RR is still
under-discussed. Therefore, it is timely to insightfully discuss the ammonia quantification
methods and their compatibility with the conditions of the sample solutions obtained from
NRR or NO3 − RR works.
In this focused review, we summarize the state-of-the-art ammonia quantification
methods, emphasizing the advantages, limitations, and practicality for NRR and NO3 − RR.
We introduce fundamental knowledge for a better understanding of each method. We also
provide our perspective on the considerations for selecting the suitable detection methods
based on the sample solution’s condition and for conducting the measurement process.
Therefore, a proper ammonia quantification procedure can be designed, which makes the
results more convincing and reliable.

2. Ammonia in Solution
Ammonia is a polar molecule that can dissolve in water due to its high solubility
(482 g L−1 at 24 ◦ C). In an aqueous solution, ammonia can simultaneously exist in two
forms of species, either in the form of unionized ammonia (NH3 ) or ammonium ion (NH4 + ).
These two species are in equilibrium (Equation (1)) and the equilibrium can be shifted to a
specific species by changing the temperature or pH value. As shown in Equations (2) and
(3), the fraction of unionized NH3 increases with increasing temperature and pH.

NH3(aq) + H2 O(l ) ↔ NH4+ (aq) + OH− (aq) (1)

pKa = 0.09018 + 2729.92 (273.15 + T)−1 (2)

  −1
Fraction of unionized NH3 = 100 1 + 10pKa −pH (3)

where pKa is the dissociation constant of the NH4 + ion and T is the temperature (◦ C) [31]. At
25 ◦ C, NH4 + ions are the predominant species at a pH lower than 9.25, while the unionized
NH3 becomes a dominant species at a pH higher than 9.25 (Figure 2) [37–39]. Because of
this property, the determination of ammonia is mostly performed in the aqueous solution.
In this case, a certain amount of acid or alkaline solution can be added to the solution to
shift the equilibrium toward unionized NH3 or NH4 + ions. In NRR and NO3 − RR, either
unionized NH3 or NH4 + can be used to represent the total ammonia production rate.

Figure 2. The molar fraction of NH4 + ions and unionized NH3 as a function of pH. Adapted from
Ref. [39] Copyright 2022, MDPI.
Energies 2023, 16, 27 4 of 22

3. Ammonia Quantification Methods

There are several methods and analytical instruments that can be used for ammonia
quantification. These include, but are not limited to, spectrophotometric methods, IC, ISE,
fluorometric, enzymatic, conductivity, and titrimetric methods. Among these methods,
spectrophotometric methods are widely used for ammonia quantification in NRR and
NO3 − RR due to their high accuracy, low detection limits, reproducibility, and simplicity.
In recent years, the IC method and 1 H NMR have also been used. Combining with 15 N2
isotopes as a feed gas during the NRR, the 1 H NMR method can verify the origin of the
produced ammonia. In the following discussion, ammonia quantification methods will be
discussed in detail, focusing on the spectrophotometric methods, IC, 1 H NMR, ISE, and fluo-
rometric methods as the major quantification methods used in NRR. Meanwhile, additional
methods will also be introduced to give a broader perspective on ammonia quantification.

3.1. Spectrophotometric Method

The spectrophotometric method is a facile and inexpensive analytical technique to
measure the absorbance or transmittance of light. In ammonia-related fields, this method
was initially used for ammonia quantification in water quality control [33]. Today, this
method is also widely used for ammonia quantification in NRR and NO3 − RR because it
has high accuracy, low detection limits, and good repeatability [15,40].
The spectrophotometric method involves the absorbance of ultraviolet-visible (UV-
vis) light by molecules. The UV light covers wavelengths from 100–380 nm, while the
visible light ranges up to 800 nm. Most UV-vis spectrophotometers possess a working
wavelength from 200–1100 nm. However, in many ammonia-related fields, including NRR
and NO3 − RR, the practical wavelength ranges from 200 to 800 nm, in which the specific ab-
sorption wavelengths used for quantification vary depending on the color of the compound
in the solution. In this regard, compounds having a conjugated unsaturated covalent bond
that can absorb UV-vis light at a particular wavelength are called chromophores, and they
confer color of the substance. Water and alcohol are considered excellent media for UV-vis
measurements since they are transparent and do not absorb UV-vis light. In comparison,
when dealing with compounds that are not dissolved in water or alcohol, acetone and
dimethylformamide (DMF) can be alternative solvents. However, both acetone and DMF
absorb light below 320 and 275, respectively [41].
UV-vis spectroscopy is based on the electronic transition of molecules that absorb
light [42]. The light absorption results in the excitation of electrons from the ground state
or lower energy orbital (highest occupied molecular orbital, HOMO) to the excited state
or higher energy orbital (lowest unoccupied molecular orbital, LUMO). The energy for
the absorbed wavelength is equal to the energy gap between HOMO and LUMO. The
relationship between the light absorption with the concentration of molecules is correlated
by the Beer-Lambert law, as shown in Equation (4).

I0
A = log =εcl (4)
I
For a given wavelength, A is the light absorbance, which is proportional to the l, length
of sample cell (cm), c, molar concentration of the solute, and ε, molar absorptivity, which is
specific for every compound. During the UV-vis measurement, the light intensity of the
sample solution, I, is measured with respect to the intensity of the reference, I0 [43]. The
concentration of the sample solution is then determined after calibration using a series of
standard solutions [41]. Notably, the l and ε are the same between the sample solution and
the standard solutions used for calibration.
Technically, a UV-vis spectrophotometer directs light to a sample, and the transmitted
light is detected by the detector on the opposite side (Figure 3a). The transmittance indicates
the portion of light that is being absorbed by the sample at each wavelength. The absorbance
values are then plotted (y-axis) as the function of wavelength (x-axis) in a UV-vis spectrum.
The wavelength with the highest peak (λmax ) is typically used for quantification [41,42].
 Technically, a UV-vis spectrophotometer directs light to a sample, and the
transmitted light is detected by the detector on the opposite side (Figure 3a). The
transmittance indicates the portion of light that is being absorbed by the sample at each
wavelength. The absorbance values are then plotted (y-axis) as the function of wavelength
(x-axis) in a UV-vis spectrum. The wavelength with the highest peak (λmax) is typically
Energies 2023, 16, 27 5 of 22
used for quantification [41,42]. Spectrophotometric ammonia determination can be
divided into two specific methods, i.e., indophenol blue method and Nessler’s method,
which will be discussed in detail in the following sections.
Spectrophotometric ammonia determination can be divided into two specific methods, i.e.,
indophenol
3.1.1. blue Blue
Indophenol method and Nessler’s method, which will be discussed in detail in the
Method
following sections.
The indophenol blue method is based on the reaction of ammonia with hypochlorite
and
3.1.1.phenol in an Blue
Indophenol alkaline
Methodcondition, which produces blue-colored indophenol. This
reaction is called the Berthelot reaction, and because it involves the use of phenol, the
The indophenol blue method is based on the reaction of ammonia with hypochlo-
indophenol blue method is also known as the phenate method. The reaction in the
rite and phenol in an alkaline condition, which produces blue-colored indophenol. This
indophenol blue method proceeds via several steps, including the reaction between
reaction is called the Berthelot reaction, and because it involves the use of phenol, the
ammonia and hypochlorite at pH 9.7–11.5 as the first step. This reaction produces
indophenol blue method is also known as the phenate method. The reaction in the in-
monochloramine, which further reacts with the phenol, producing quinone chloramine.
dophenol blue method proceeds via several steps, including the reaction between ammonia
The formation of quinone chloramine is facilitated by the addition of sodium
and hypochlorite at pH 9.7–11.5 as the first step. This reaction produces monochloramine,
nitroprusside
which furtheror sodium
reacts with nitroferricyanide as a quinone
the phenol, producing catalyst. chloramine.
Subsequently,
Thethe quinone
formation of
chloramine further reacts with phenol to form indophenol, which dissociates
quinone chloramine is facilitated by the addition of sodium nitroprusside or sodium in theni-
alkaline solutionasresulting
troferricyanide in Subsequently,
a catalyst. a blue color. Therefore,
the quinonethechloramine
indophenol product
further canwith
reacts be
quantitatively
phenol to form determined
indophenol,using a UV-vis
which spectrophotometer
dissociates in the alkalineat a wavelength
solution of 630–655
resulting in a blue
nm. The overall process of the reaction in the indophenol blue method is presented
color. Therefore, the indophenol product can be quantitatively determined using a UV-vis in
Equation (5) [44].
spectrophotometer at a wavelength of 630–655 nm. The overall process of the reaction in
the indophenol blue method is presented in Equation (5) [44].

(5)
(5)

Modification of the indophenol blue method was then developed, specifically by

substituting the phenol
Modification of theusing sodium salicylate
indophenol blue methodor salicylic
was thenacid.developed,
This modified method is
specifically by
also known asthe
substituting thephenol
“salicylate
usingmethod”.
sodium In additionortosalicylic
salicylate being less harmful
acid. than phenol,
This modified the
method
use of salicylate
is also known ascompounds is tomethod”.
the “salicylate avoid theIn formation
additionof toabeing
harmful
lesssubstance,
harmful than suchphenol,
as o-
the use of salicylate
chlorophenol, duringcompounds
the process.is to avoid thebecause
However, formationof of
thea lower
harmful substance,
reactivity of such
the
as o-chlorophenol,
salicylate comparedduring the process.
to phenol, the However,
salicylate because
method ofrequires
the lowera reactivity
much higherof the
salicylate compared
concentration to phenol,
of salicylate the salicylate
to reach a similar method requires
sensitivity a much
as phenol. Thehigher concentration
reaction involves
of salicylate
the to reach
same reaction stepa between
similar sensitivity
the ammoniaas phenol. The reactionto
and hypochlorite involves the same reaction
form monochloramine,
step
as in between the ammonia
the original indophenol andblue
hypochlorite
method. toHowever,
form monochloramine,
in the salicylateas in the original
method, the
indophenol blue method. However, in the salicylate method,
monochloramine reacts with salicylate, producing 5-aminosalicylate. The 5- the monochloramine reacts
with salicylate, is
aminosalicylate producing
oxidized,5-aminosalicylate.
and it further reacts The 5-aminosalicylate is oxidized,
with salicylate, forming and it
a colored
further reacts
compound. Thewith salicylate,
apparent colorforming a colored
may be varied compound.
depending Theammonia
on the apparentconcentration
color may be
varied
in depending
the sample. on themay
The color ammonia
changeconcentration
from yellow to ingreen
the sample.
and thenThetocolor
blue,may
alongchange
with
from yellow
increasing to green
ammonia and then to(Figure
concentration blue, along withoverall
3b,c). The increasing ammonia
reaction is shown concentration
in Equation
(Figure 3b,c). The overall reaction is shown in Equation (6).
(6).

(6)
(6)
Energies 2023, 16, 27 6 of 22

Figure 3. (a) Schematic illustration of UV-vis spectrophotometer for double beam instrument. The
tungsten lamp and D2 lamp emit visible and ultraviolet lights, respectively. The light is directed
to the monochromator, which determines the wavelength of the sample. Adapted with permission
from Ref. [41] Copyright 2018, Wiley-VCH. Ammonia determination using indophenol blue method,
(b) UV-vis spectra of standard solutions with different concentrations, and (c) corresponding cali-
bration curve. The absorbance values for the calibration curve were obtained from a wavelength of
655 nm. Inset of (c) is the photograph of the standard solutions after 2 h of reaction with indophenol
reagent. Adapted with permission from Ref. [45] Copyright 2021 Royal Society of Chemistry. Ammo-
nia determination using Nessler’s method. (d) UV-vis spectra of the standard solution with different
concentrations, and (e) corresponding calibration curve. The absorbance values for the calibration
curve were obtained from the wavelength of 420 nm. Inset of (e) is the photograph of the standard
solution after 20 min of reaction with Nessler’s reagent. Adapted with permission from Ref. [46]
Copyright 2020, Royal Society of Chemistry.

In both the indophenol blue method and the salicylate method, citrate buffer is of-
ten used to stabilize the pH and prevent the precipitation of hardness cations, such as
magnesium or calcium ions, at a high pH [35,47]. The indophenol blue method and the
salicylate method are able to measure ammonia at low concentrations (0–0.6 mg L−1 of
NH3 -N) with good accuracy and repeatability [14]. However, it takes a relatively long
time for the samples to be ready for measurement (1–2 h). It is worth noting that most of
Energies 2023, 16, 27 7 of 22

the NRR and NO3 − RR reports employed the salicylate method to quantify the produced
ammonia, according to the reagent that they used (they used a salicylate compound in-
stead of phenol). However, they referred to their method as the indophenol blue method
instead of the salicylate method, possibly due to the origin of the salicylate method de-
rived from the indophenol blue method and the same principle between them, or just for
simplification purposes. Therefore, we will use the term indophenol blue method in the
following discussion.

3.1.2. Nessler’s Method

Nessler’s method refers to the reagent being used, i.e., Nessler’s reagent. This reagent
consists of mercury (II) iodide or potassium iodide in an alkaline solution, either in sodium
hydroxide or potassium hydroxide. Upon reaction with ammonia, the Nessler’s reagent
forms a yellow-colored compound, which can be determined using a spectrophotometer at
wavelengths of 410–425 nm. The color intensifies from yellow to yellow-orange/reddish-
brown along with an increase in ammonia concentration (Figure 3d,e) [47]. The overall
reaction is shown in Equation (7).

2K2 HgI4 + NH3 + 3KOH → HgO·Hg(NH2 )I + 7KI + 2H2 O (7)

The detection of ammonia using Nessler’s method can be interfered with by various
metal cations, especially the hardness metal cations. The metal cations may precipitate
as hydroxides in the high alkaline solution, which interfere with the spectrophotometric
measurements. The typical approach to remove the turbidity in the sample solution is by
adding zinc sulfate and sodium hydroxide, followed by filtration. However, this process is
relatively tedious and time-consuming. Alternatively, a Rochelle salt (potassium sodium
tartrate) can be put in to prevent turbidity.
Nessler’s method can detect ammonia concentrations at a comparable detection limit
to the indophenol blue method (0.025–5.0 mg L−1 of NH3 -N), but with a relatively faster
process [14]. Due to the sensitivity of this method to the reaction time, the measurement of
the colored sample using a spectrophotometer is recommended to be performed around
10–30 min after mixing the sample solution with Nessler’s reagent for an accurate result.
However, one should note that Nessler’s method employs a mercury-containing chemical,
which is toxic and harmful. Therefore, the reagent and the related samples should be
properly handled, stored, and disposed of [35,47].

3.2. IC Method
Ion chromatography is a separation method that works based on the interaction
between polar molecules or ions with the ion exchanger (stationary phase) and an eluent
(mobile phase) [48]. In NRR and NO3 − RR, the cationic exchangers, which possess a
negatively charged functional group, are used because the ammonia is detected in the
form of NH4 + ions. The cationic exchangers are embedded in the column. A low-capacity
ion-exchange column allows the use of the dilute ionic solution as an eluent. This eluent
has an adequately low conductivity background. Therefore, the conductivity of the ions
can be directly measured. This technique is known as the non-suppressed IC. Meanwhile, if
the eluent is diluted so it converts to a neutral form after the separation, then the technique
is called suppressed IC. Both techniques demonstrate high sensitivity up to mg L−1 level
with the ability to detect multiple ions within minutes [49].
The primary equilibrium in the IC is the ion-exchange displacement of an eluent ion (E)
that is initially attached to the stationary phase (denoted by the subscript r) by an analyte
(A) in the mobile phase (denoted by subscript m). The equilibrium for cation exchange of a
single charged ion is shown in Equation (8).

A+ + + +
m + Er ↔ Em + Ar (8)
Energies 2023, 16, 27 8 of 22

The charge of the cation is balanced by anions (of the same charge as cations) in the
mobile phase. However, this anion does not involve the cation exchange process. The
common eluent for cation exchange is H+ , coming from sulfuric acid, citric acid, nitric
acid, or tartaric acid. Specifically, for suppressed IC, sulfuric acid or methanesulfonic acid
(MSA) is usually used. In IC, the selectivity is mostly altered by modifying the nature of the
stationary phase or by just using the suitable stationary phase. In this case, the selectivity
in IC can be described as the tendency of the exchange process between the ion in the
analyte of interest, and the eluent ion in the stationary phase. Cation exchange selectivity is
commonly altered using different types of functional groups, such as sulfonate, carboxylate,
and phosphate [49].
A typical IC instrument includes a pump, an injector, a column, a suppressor, and
a recorder or data system (Figure 4a). Ion exchange separation mainly occurs in the
column packed with an ion exchanger, which is commercially available. When the samples
containing certain cations are injected into the chromatography instruments, the cations
flow with the eluent through a column. The speed of the cations flowing through the
column differs depending on their interaction with the ion exchanger, and they are then
separated. The conductivity detector is then usually used to detect the separated cation,
which results in a series of peaks with different retention times. Each cation is identified
based on its retention time, and the area of the peak represents the concentration of the
cation (Figure 4b). In this case, the peak area is proportional to the concentration of the
cation. By comparing the obtained peak area with the standard calibration curve, the
concentration of the cation can be quantified (Figure 4c,d).

Figure 4. (a) Schematic diagram of standard IC instrument. Adapted from Ref. [50] Copyright 2017,
Elsevier. (b) High-speed separation of nine common cations at 27 ◦ C. Experiment was performed
using Dionex CS-12A cation exchange column (carboxylate-functionalized cation-exchange column),
eluent: 17 mM MSA at 0.5 mL/min, 20 µL injection, 50 µM analyte concentration. Adapted with
permission from Ref. [51] Copyright 2003, Elsevier. (c) Chromatogram of NH4 + standard solutions
with various concentrations and (d) corresponding calibration curve. Adapted with permission from
Ref. [45] Copyright 2021, Royal Society of Chemistry 2021.
Energies 2023, 16, 27 9 of 22

Compared to the spectrophotometric method, the IC method has some advantages. First,
the IC method can measure NH3 -N concentrations ranging from 0.02 to 40 mg L−1 [14,25].
Moreover, in a single operation process, it can simultaneously detect multiple cations with
high sensitivity and repeatability. Second, it has high selectivity because it can quantify
inorganic and organic cations by adjusting the parameter and operational procedure.
Third, many types of cations can be detected and quantified in one running process [35].
Unfortunately, the peaks originating from the cations with closed retention times may
overlap with each other, which interferes with the detection. For example, the peaks of
NH4 + ions may overlap with the peaks of Na+ , K+ , and Li+ , which limits its application
for NH4 + quantification in electrolytes containing those ions [14,47]. Therefore, other
approaches are needed to improve the resolution of the peaks; for example, by using a
column with a higher cation-exchange capacity or applying a column-switching technique
to improve the separation of NH4 + and Na+ peaks [31].

3.3. 1 H NMR Method

Many nuclear isotopes, including 1 H, 14 N, and 15 N, are magnetically active with non-
zero spin and the NMR instruments measure the radio frequency (RF) associated with those
nuclei. Similar to other spectroscopy methods, the NMR analyzes the structure or chemical
composition of molecules based on their absorption of electromagnetic radiation. In this
case, the NMR specifically works at the radio-wave frequency, which affects a transition in
the nuclear spin level.
Many atomic nuclei have a property called spin, by which we can illustrate as if
they are spinning. The energy of spin states is not equal in an applied magnetic field.
Because nuclei are charged particles, they have their own magnetic moments (µ), which are
generated by their charge and spin. In an applied magnetic field, there are two magnetic
moment alignments of nuclei. The alignment can be in the same direction (+ 12 spin state) or
in the opposite direction (− 12 spin state) relative to the magnetic field direction. The + 12 spin
state has a lower energy level [43]. The resonance occurs when the nuclei with the same
alignment as the applied magnetic field absorb energy, which changes their orientation
(Figure 5a). The absorbed energy must be equal to the difference between the two states, as
shown in Equation (9).
 
Eabsorbed = E− 1 state − E+ 1 state = hν (9)
2 2

where Eabsorbed is the energy absorbed by the nuclei, E− 1 state is the energy of − 12 spin state
2
(opposite direction against the magnetic field direction), E+ 1 state is the energy of + 12 spin
2
state (same direction as the magnetic field direction), h is the Planck constant, and ν is the
frequency of the electromagnetic radiation [43].
Energies 2023, 16, 27 10 of 22

Figure 5. (a) Absorption process in NMR. (b) Schematic representation of a typical NMR spectrometer.
Adapted with permission from Ref. [52] Copyright 2019, Georg Thieme Verlag KG. (c) 1 H NMR
spectra of 14 NH4 + and 15 NH4 + produced from NRR reaction using 14 N2 and 15 N2 isotopes. Adapted
with permission from Ref. [45] Copyright 2021, Royal Society of Chemistry. (d) 1 H NMR spectra
of 15 NH4 + standard solutions with various concentrations and (e) corresponding calibration curve.
Adapted with permission from Ref. [53] Copyright 2020, American Chemical Society.

In practice, energy difference in the energy levels (and therefore, the nuclei population)
is a function of the applied magnetic field. The stronger the magnetic field, the higher the
energy difference between the possible spin states. In an applied magnetic field, the nuclei
precess in their axis with various alignments. The frequency at which a nucleus precess is
proportional to the strength of the applied magnetic field. In this case, each isotope has its
characteristic frequency. For example, in a 7.5 T of magnetic field, the 1 H nucleus precesses
at 300 MHz, while at 21.1 T, 1 H precesses at 900 MHz [54].
The resonance occurs when external electromagnetic radiation matches with the
nucleus precession. As also shown in Figure 5a, during this resonance process, the spin
alignment of the nuclei changes from + 12 spin state to − 12 spin state, which increases the
population of higher energy spin states. This process continues until the population of the
high energy spin states is equal to the lower energy spin states. This condition is called
saturation, which should be avoided as no net signal can be observed. At this stage, external
electromagnetic radiation is turned off. Therefore, the resonance stops. With cessation of
resonance, the system changes back to the initial condition, where the high energy spin state
(− 12 ) nuclei relax to the lower energy (+ 12 ) spin state. The relaxation process is accompanied
by RF emission. Therefore, the detector can record the intensity of RF from each nucleus.
This process is reiterated, which strengthens the signal from each nucleus and reduces
the noise. The software then calculates peak properties, such as chemical shift, intensity,
multiplicity, and J-coupling (Figure 5b) [43,54].
In NRR and NO3 − RR, 1 H NMR is mostly used, instead of 15 N NMR, to detect 15 NH3
because of the extremely low gyromagnetic ratio of 15 N. In this case, 1 H NMR can differen-
tiate the 15 NH4 + and 14 NH4 + because 14 NH4 + is a spin-1 nucleus and 15 NH4 + is a spin- 12
nucleus. Therefore, the coupling between 1 H and 15 N in 15 NH4 + will generate a doublet
Energies 2023, 16, 27 11 of 22

signal with a spacing of 73 Hz. Meanwhile, the coupling between 1 H and 14 N in 14 NH4 +
results in a triplet signal with a spacing of 52 Hz (Figure 5c) [36,47,53].
Measurement using 1 H NMR in NRR usually aims to confirm the origin of the pro-
duced ammonia. Before 1 H NMR detection, the NRR experiments are first performed by
using the 15 N2 as an N source instead of 14 N2 . Therefore, the detection of 15 NH3 in NMR
spectra verifies that the produced ammonia is originated from N2 gas instead of other
impurities. In addition to confirming the origin of ammonia qualitatively, more recently, 1 H
NMR was also used for 15 NH3 quantification, as reported by Jang et al. For this purpose,
measurements of a series of standard solutions were performed. The peak area of the 1 H
NMR spectra was then plotted as a function of concentration to obtain the calibration curve
(Figure 5d,e). Prior to measurements, the sample solutions were acidified to reach a pH of
∼3 by adding HCl to convert 15 NH3 to 15 NH4 + [53].
The 1 H NMR method offers high sensitivity, appreciable repeatability, and straight-
forward discrimination against the contaminant ammonia [34]. Furthermore, it does not
need any advanced chemical manipulation, as required in the spectrophotometric method.
However, it requires complex and expensive instrumentation.

3.4. ISE Method

There are typically two types of electrodes that can be used to measure the dissolved
ammonia; i.e., an ammonium ion-selective electrode and an ammonia gas sensing electrode.
As its name suggests, the ammonium ion-selective electrode (NH4 + -ISE) measures NH4 +
ions dissolved in the solution. It measures the potential difference between the reference
electrode and the ion electrode (Figure 6a) [55]. This electrode is equipped with an NH4 +
ion-specific membrane, typically a polyvinylchloride (PVC) membrane. Before performing
ammonia detection using NH4 + -ISE, the sample solution is acidified to convert NH3(aq) into
NH4 + ions. When the electrode is placed into the sample solution, the potential in the ion
electrode develops against a reference electrode (typically Ag/AgCl) [56]. The value of the
potential is proportional to the concentration of the NH4 + ion in the solution, in accordance
with the Nernst equation (Equation (10)). The concentration of NH4 + can be quantified
using the calibration curve.
RT
E = E0 + ln Q (10)
zF
where E is the measured voltage (V), E0 is the reference constant (V), R is the universal
gas constant (J mol−1 K−1 ), T is the temperature (K), z = charge of ion, F is the Faraday
constant (C mol−1 ), and Q is the reaction quotient of the cell reaction [57].

Figure 6. Schematic diagram of (a) NH4 + ion selective electrode. Adapted with permission from
Ref. [57] Copyright 2019, Royal Society of Chemistry. (b) A typical membrane-based electrochemical
gas sensor. For the ammonia gas sensing electrode, the pH electrode is typically used as a selective
electrode. Adapted from Ref. [58] Copyright 2017, Elsevier.
Energies 2023, 16, 27 12 of 22

NH4 + -ISE has a wide range of detection limits: 0.06–20,000 mg L−1 NH3 -N (Radiome-
ter Analytical ISE25NH4-9) [14]. This value varies for each commercial product. The major
limitation of NH4 + -ISE is disturbance from the cations, which have similar behavior to ion
electrodes, such as Na+ and K+ . Hence, the other cations can be detected as ammonium,
which results in a false positive. The interference by the other cations can be quantified
based on the cross-sensitivity ratio, as shown in Equation (11).
c M+
c NH4 + = (11)
CSR

where CNH4 + is the NH4 + ions concentration (mg L−1 ), c M+ is the concentration of interfer-
ence cation, and CSR is the cross-sensitivity ratio. The CSR value for K+ is 1:(15–30) against
NH4 + . This CSR value means that 20 mg L−1 of K+ ions will have the same potential
difference as ∼1 mg L−1 NH4 + . Meanwhile, Na+ has a CSR value of 1:(1000–1300) against
NH4 + . The interference of Na+ to NH4 + is estimated in the same way as K+ [57].
The ammonia gas-sensing electrode measures the dissolved ammonia in the form
of unionized NH3 . It has a wide detection range of 0.01–17,000 mg L−1 NH3 -N (Orion
9512HPBNWP) and the detection range can be varied in different commercial products [14].
The electrode consists of a pH electrode, a hydrophobic gas-permeable membrane, and
a reference electrode (Figure 6b) [58]. It typically uses ammonium chloride solution as
an internal solution. The gas permeable membrane separates this internal solution to the
sample solution. Prior to ammonia detection, a strong base is added to the sample solution
to reach a pH value higher than 11. Therefore, the dissolved NH4 + ions in the sample
solution are converted into dissolved NH3(aq) , which then diffuse through the membrane
to the internal solution until the NH3 partial pressure of both the sample solution and the
internal solution reaches equilibrium. The diffusion of NH3(aq) from the sample solution to
the internal solution changes the pH of the internal solution, and these pH changes can be
detected using a pH electrode [14,31]. In this regard, the pH change is proportional to the
concentration of the NH3(aq) in the sample solution [44]. However, because the gas-sensing
electrode detects the ammonia in the form of dissolved ammonia gas (NH3(aq) ), there is
a possibility that the NH3(aq) can be released from the sample solution. Therefore, it is
recommended to have a good sealing between the internal solution and sample solution,
or to use a closed container with a small opening to tackle the NH3(aq) releasing problem.
Moreover, the performance of the ammonia gas sensing electrode is highly affected by
the concentration of dissolved ammonia and the dissolved ions. It slowly works in a low
concentration of ammonia. However, turbidity or color stability, which can be problems
in the spectrophotometer method, do not influence the performance of the gas-sensing
electrode [31].
Both NH4 + -ISE and the NH3 gas-sensing electrode provide a facile and rapid process
for ammonia determination. They are also able to measure ammonia in a much wider range
of concentrations. However, their detection accuracy, precision, and repeatability in low
ammonia concentrations (i.e., <0.5 mg L−1 NH3 -N) is lower than in higher concentrations.
Furthermore, because of the deterioration of the membrane, the lifetime of the ISE is limited
to between months to a year [14].

3.5. Fluorometric Method

The fluorometric method offers high sensitivity for ammonia detection at nanomolar
concentrations (1.0 × 10−4 –2.8 × 10−3 mg L−1 NH3 -N) [14]. This method is based on the
fluorescence process of molecules. When molecules are subjected to light, the molecules
absorb light (excitation), which changes their electronic state from the ground state (S0 ) to
one of the vibrational levels of the excited electronic state (S1 ). The fluorescence process
occurs when the electron relaxes from the singlet excited state (S1 ) to the singlet ground state
(S0 ), which is accompanied by light emission (Figure 7a) [59]. The lifetime of fluorescence
is very short (1 × 10−9 to 1 × 10−7 s). The energy (or wavelength) of the emitted light
from the fluorescence process is determined by the energy difference between the ground
Energies 2023, 16, 27 13 of 22

state (S1 ) and the singlet excited state (S1 ), and is also influenced by the energy lost due to
vibrational relaxation. This relationship is shown by Equation (12).

EFluor = E Abs − EVib − ESolv.relax (12)

where EFluor is the energy of emitted fluorescence, E Abs is the absorbed energy, EVib is the
vibrational relaxation energy, and ESolv.relax is the energy associated with the solvent cage
of molecules to reorient itself in the state. Moreover, due to the energy loss, the wavelength
of the emitted fluorescence shifts to a lower energy (longer wavelength) than the excitation
wavelength (Stokes shift) [60].
Ammonia detection using the fluorometric method employs o-phthaldialdehyde (OPA)
and sulfite as the reagents, which react with ammonia forming a highly fluorescent isoin-
dole derivative (Equation (13)) [61–64]. The fluorescent product has maximum excitation
and emission wavelengths at 362.5 and 423.0 nm, respectively (Figure 7b) [63,65]. The
fluorescence detector is then used to detect the emitted fluorescence signal, which is pro-
portional to the concentration of ammonia.

(13)

Practically, in a typical flow injection fluorescence system (Figure 7c), the sample is
injected to the sample loops, where the carrier facilitates the sample to react with OPA and
sulfite, producing fluorescence products in the reaction coil. The product is then carried
to the detector [66]. Note that the presence of amines and amino acids in the solution
interferes with the ammonia detection, since both compounds may also react with the OPA,
producing fluorescence products [62]. To avoid such interference, a purge-and-trap strategy
can be helpful. In this technique, the sample solution is added with sodium hydroxide
to convert all NH4 + ions to NH3(aq) . Subsequently, the solution is purged with argon gas
to release the NH3(aq) . The released NH3 is then trapped in a hydrochloric acid solution.
The NH4 + concentration in the hydrochloric acid solution is then determined using the
fluorometric method [66].
In NRR or NO3 − RR works, where the reaction does not involve the use or the forma-
tion of amine or amino acid-related compounds, the interference from amines and amino
acids can be neglected. Because of its sensitivity, the fluorescence method can be considered
for ammonia detection in NRR because the produced ammonia is still often very limited
(at micromolar level). However, for NO3 − RR, which typically produces a more significant
amount of ammonia (at the millimolar level), the fluorometric method may not be suitable
for ammonia detection because too much dilution is needed to fit the method’s linearity
ranges, which may lead to errors.

3.6. Enzymatic, Conductivity, and Titrimetric Methods

In addition to the aforementioned five major ammonia quantification methods, there
are some other methods that are also known, i.e., the enzymatic method, the conductivity
method, and the titrimetric method [31]. However, these methods are rarely used at the
present time, likely due to sensitivity issues and complex procedures. In this following
discussion, the three methods are briefly introduced.
Energies 2023, 16, 27 14 of 22

Figure 7. (a) Jablonski diagram. S is the singlet states (paired spin), and T is the triplet states (unpaired
spin). Solid lines represent radiative processes, while dashed lines represent nonradiative processes.
Adapted with permission from Ref. [60] Copyright 2019, Wiley VCH. (b) Excitation and emission
spectra from the product of ammonia-o-phthaldialdehyde-sulfite. Adapted with permission from
Ref. [65] Copyright 2014, Hindawi. (c) Schematic diagram of the flow injection-fluorescence system.
Adapted with permission from Ref. [66] Copyright 2016, Taylor & Francis Publisher.

The first method is the enzymatic method, which uses glutamate dehydrogenase
enzyme to catalyze the reaction of 2-oxoglutarate and reduced nicotinamide adenine
dinucleotide (NADH) in the presence of ammonia. Specifically, the enzyme catalyzes the
reductive amination of 2-oxoglutarate, where NADH is oxidized during the glutamate
formation from the ammonia present (Equation (14)). Therefore, the concentration of
ammonia is determined based on the decrease of NADH concentration, which can be
spectrophotometrically detected by the change of the NADH absorbance peak at 340 nm.
However, this method is highly vulnerable to pH and temperature [31,67–70].

NADH + NH4 + + 2-oxoglutarate → L-glutamate + NAD+ + H2 O (14)

The second is the conductivity method, which is based on changes in solution conduc-
tivity due to the formation of salt as a result of the ammonia reaction with the acid solution.
In this process, the gas containing ammonia is flowed into the sulfuric acid solution. The
ammonia will react with the sulfuric acid, producing salts that decrease the solution’s
conductivity. The change in conductivity is then monitored using a conductivity meter.
The decrease in conductivity is proportional to the amount of ammonia [71]. Despite its
simplicity, this method is prone to interferences because conductivity is not only specifically
influenced by ammonia. The presence of other ions also changes conductivity. Therefore,
impurities originating from the samples, feed gas, instruments, or side products may easily
deviate from ammonia detection [31].
Energies 2023, 16, 27 15 of 22

The last method is the titrimetric method, which is usually used for the detection of
ammonia with a concentration higher than 5 mg L−1 . In this method, the pH of the sample
solution is increased to 9.5 to convert NH4 + ion into NH3(aq) . Then, the distillation process
is performed on the sample solution to release all the NH3(aq) , in which the distillate is
accommodated in a container containing boric acid solution and an indicator. Titration
using a standard sulfuric acid solution is then performed on the distillate solution to obtain
ammonia concentration. This process requires the measurement of blank samples for
control experiments [31,72]. However, the requirements for the distillation process using
this method may make this method less practical.

3.7. Comparison of Ammonia Quantification Methods and Their Application in Nitrogen/Nitrate

Reduction Reaction
A comparison of the five major ammonia detection methods is presented in Table 1
and is illustrated in Figure 8. In Table 1, we also provide some representative catalysts,
together with their activity (ammonia yield, in mg L−1 NH3 -N), to represent the current
ammonia production levels in NRR and NO3 − RR. Among the five ammonia detection
methods, most NRR or NO3 − RR studies employed spectrophotometric methods as the
main ammonia detection method. The IC and 1 H NMR methods have also been used.
The isotope labeling experiment using 1 H NMR has been mostly used to qualitatively
verify the origin of the produced ammonia. The ISE and fluorescence methods have been
mostly utilized in water quality control. Here, the fluorometric method showed the lowest
detection limit, which could detect up to the nanomolar level. Fortunately, there have been
many reagents and kits for ammonia quantification that are now commercially available.
These commercial products can potentially simplify experimental procedures. Readers are
directed to the excellent review paper that provided the list of those commercial products
for further information [31].

Figure 8. Illustration of five major quantification methods that have been used or can potentially be
used for ammonia quantification in nitrogen/nitrate reduction reactions.
Energies 2023, 16, 27 16 of 22

Table 1. Comparison of the ammonia quantification methods with some representative catalysts.

Detection Range NH3 -N Yield

Method Remarks Representative Samples Reaction System Ref.
(mg L−1 NH3 -N) (mg L−1 )
(a)
Bi nanosheet EC-NRR 0.14 (−0.8 V) [73]
Cu/oxygen deficient-TiO2 EC-NRR 0.06 (−0.5 V) (a) [28]
(a),(b)
Advantages: Bi2 MoO6 EC-NRR 0.08 (−0.6 V) [74]
High accuracy and repeatibility, simple procedure and instrumentation, stable Bi4 O5 Br2 /ZIF-8 PC-NRR 0.46 [75]
Spectrophotometer method:
0–0.6 color, low toxicity.
Indophenol blue method EC-
(salicylate method) Cu/Cu2 O 171.4 (−0.85 V) (a),(c) [30]
Limitations: NO3 − RR
Time-consuming, sacrificial reagent interference.
EC-
Defected CuO 364.0 (−2.2 V) (a),(b),(c) [76]
NO3 − RR
TiO2 (P25) PC-NO3 − RR ∼2.3 [77]
(a)
Bi2 Te3 EC-NRR 0.2 (−0.4 V) [78]

Advantages: Bi nanocrystal EC-NRR 4.6 (−0.8 V) (a),(b) [69]

High accuracy and repeatability, simple procedure and instrumentation, C3 N4 /Bi2 MoO6 PC-NRR 1.8 [79]
timesaving.
Spectrophotometer method: 0.025–5.0 Bi2 O2 CO3 PC-NRR 13.4 [80]
Nessler’s method
Limitations:
Metallic ion interference, pH effect, sacrificial reagent effect, toxicity. GQDs/Bi2 WO6 PC-NRR 0.3 [81]
EC- 1675.6 (−0.24 V)
CuO@Co3 O4 [82]
NO3 − RR (a),(b),(c)

Advantages:
Detection of wide range of concentrations, high selectivity and precision,
rapid detection. Ag/AgI-δ-Bi2 O3 PC-NRR 3.3 [83]
Ion chromatography 0.02–40
Limitation:
Overlapped peak between NH4 + with Na+ , K+ , or Li+ . B-doped Bi (photoanode) PEC-NRR 0.03 (+0.48 V) (a),(b) [84]
Advantages:
High sensitivity, straighforward discrimination of 14 NH4 and 15 NH3 ,
1 appreciable repeatibility.
H NMR / Defected Bi nanoplate EC-NRR 0.11 (−0.9 V) (a),(b) [85]
Limitation:
Complex and expensive instrumentation.
Advantage:
Rapid detection
ISE method:
0.06–20,000 (d) / / / /
NH4 + -ion selective electrode Limitations:
Na+ and K+ interference, less precise and accurate at low ammonia
concentration.
Energies 2023, 16, 27 17 of 22

Table 1. Cont.

Detection Range NH3 -N Yield

Method Remarks Representative Samples Reaction System Ref.
(mg L−1 NH3 -N) (mg L−1 )
Advantage:
Rapid detection
ISE method:
0.01–17,000 (e) / / / /
NH3 gas sensing electrode
Limitations:
Escape of NH3 gas, less precise and accurate at low ammonia concentration.
Advantage:
High sensitivity (up to nanomolar level)
Fluorometric method 1.0 × 10−4 –2.8 × 10−3 / / / /
Limitations:
Reaction completeness, complex instrumentation, less suitable for high
ammonia concentration.
(a)V vs. reversible hydrogen electrode (RHE). (b) Because of the lack information of the volume used for calculation in the original paper, this value is calculated by assuming the volume
of the electrolyte is 40 mL, as in typical H-cells. This aims to approximate of the concentration range of the produced ammonia. (c) Involve dilution of the sample solution. (d) Radiometer
Analytical ISE25NH4-9. (e) Orion 9512HPBNWP.
Energies 2023, 16, 27 18 of 22

4. Perspective for Selecting Method and Performing Ammonia Quantification

The reaction conditions that are usually employed either in EC, PC, or PEC NRR
(and NO3 − RR), such as concentrations of produced ammonia, pH, cations, and sac-
rificial reagents, can influence detection accuracy. The indophenol blue method, the
Nessler’s method, and the IC method show high accuracy in ammonia concentrations
below ∼0.41 mg L−1 NH3 -N. However, above this concentration, the indophenol blue
method shows a positive bias [35]. Therefore, dilution is necessary for the sample solution
containing concentrated ammonia, where it will be measured using the indophenol blue
method to achieve high accuracy.
Recently, Zhao et al. also reported that, at low ammonia concentrations (<0.16 mg L−1
NH3 -N), the indophenol blue method exhibited high accuracy over a wide range of pH.
Meanwhile, the Nessler’s method worked well in neutral pH. In acidic conditions, such as in
H2 SO4 solution or electrolyte, the IC method is a good option considering less interference
compared to the spectrophotometric method [34]. However, in the presence of Na+ or K+ ,
the NH4 + peaks may overlap with the Na+ or K+ peaks; therefore, the IC instrument needs
to be optimized first before performing the ammonia detection to obtain a better resolution
of each ion peak.
In the PC-NRR (and NO3 − RR), which typically employ sacrificial reagents such as
methanol, the indophenol blue and Nessler’s methods showed deviations (Figure 9), pos-
sibly due to complex formations with ammonia [35]. Therefore, one can consider using
the IC method for ammonia detection when working with sacrificial reagents. The 15 NH3
isotope labeling or 14 NH3 measurement using 1 H NMR shows excellent stability and re-
producibility in various electrolytes. However, it requires sophisticated and expensive
instruments. Moreover, quantification of low-concentration ammonia dissolved in concen-
trated electrolytes is still challenging. As an alternative, the fluorometric method can be
employed in low ammonia concentrations, such as in EC-, PC-, and PEC-NRR. Meanwhile,
the ISE method can be a practical method for ammonia detection in a system that produces
a large amount of ammonia, such as in PC- and EC-NO3 − RR. Note that the spectrophoto-
metric method and the fluorometric method involve a reaction between the ammonia and
the reagents, where the reaction between the reagent and the ammonia is concentration
dependent. Underestimation of ammonia concentrations may occur due to incomplete
reactions. Therefore, a specified concentration of reagents and a strict time of analysis are
crucial to ensure the completeness of the reaction.

Figure 9. Photograph of the various ammonia solutions in the presence of 40% volume of methanol
as the sacrificial reagent and its derived oxidation products determined by (a,c) Nessler’s method
and (b,d) indophenol blue method. The ammonia concentration was 600 µg L−1 in all experiments.
Adapted with permission from Ref. [35] Wiley-VCH 2021.
Energies 2023, 16, 27 19 of 22

Regardless of the methods and instruments being used, it is recommended to prepare

the calibration curve from the standard solutions which closely mimic the real condition
of the sample solution; e.g., use the same solvent to prepare the standard solution as
in the real reaction, add an equivalent amount of sacrificial reagent, or adjust the pH.
Therefore, possible interference from the solution background can be minimized. The
quantification of the blank solution (or solution taken before the reaction is started) is also
highly recommended to correct the measurement results of the sample solution after the
reaction. Finally, it is also highly recommended to verify the results of one method with
other quantitative methods (perform two separate ammonia quantification with different
methods from the same sample solution) to make the ammonia quantification more reliable.

5. Conclusions
An overview of the current methods for ammonia quantification, either those that
have been used or can potentially be used for present NRR and NO3 − RR studies and in the
future, was provided. In general, the selection of ammonia quantification methods should
consider the condition of the sample solution and the predicted ammonia concentration.
The sensitivity or detection limits of the selected quantification method should meet the
range of ammonia concentrations. Moreover, possible interferants in the solution, such
as cations, sacrificial reagents, and pH, must also be considered. Although the list of
quantification methods described here is not exhaustive, this focused review is expected to
provide a guide by which to choose the most suitable ammonia quantification method in
NRR and NO3 − RR studies and properly perform measurements. Therefore, the obtained
ammonia values will be more convincing and reliable.

Author Contributions: Conceptualization, Y.H.N., writing—original draft preparation, W.P.U.;

writing—review and editing, W.P.U., H.W. and Y.H.N., supervision, H.W. and Y.H.N. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by Hong Kong Research Grant Council (RGC) General Research
Fund (GRF) CityU11305419, CityU11306920, CityU11308721 and the General Program of Science and
Technology Innovation Committee of Shenzhen Municipality JCYJ20190808181805621.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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