Available online at www.sciencedirect.
com Current Opinion in
ScienceDirect
Recent efforts to increase greenness in
chromatography
Patricia I. Napolitano-Tabares1, Isaac Negrín-Santamaría1,
Adrián Gutiérrez-Serpa1,2 and Verónica Pino1,2
Conventional high-performance liquid chromatography (HPLC)
and gas chromatography (GC) are the most widely used
analytical tools in the analytical chemistry field. However, these
successful techniques present a number of unfriendly envi-
ronmental issues. Thus, conventional HPLC needs large
amounts of organic solvents and generates high amounts of
wastes, conventional GC mostly uses non-renewable and
expensive gases such as He (particularly when combined with
mass spectrometry), and conventional HPLC and GC are both
time- and high energy-demanding. This review describes
recent modifications (2018–2020) introduced in HPLC and GC
techniques to improve their greenness, covering topics such as
mobile phases and mobile phase additives, novel stationary
phases, and mostly the miniaturization of the equipment with
the purpose of simplification and costs reduction.
Addresses
1 such as acetonitrile. Thus, HPLC improvements from a
Departamento de Química, Unidad Departamental de Química
Analítica, Universidad de La Laguna (ULL), La Laguna, Tenerife,
38206, Spain
2
Unidad de Investigación de Bioanalítica y Medioambiente, Instituto
Universitario de Enfermedades Tropicales y Salud Pública de Cana-
rias, Universidad de La Laguna (ULL), La Laguna, Tenerife, 38206
Spain
Corresponding author: Pino, Verónica (veropino@ull.edu.es)
Current Opinion in Green and Sustainable Chemistry 2021,
32:100536
This review comes from a themed issue on Green Analytical
Chemistry
Edited by Mihkel Koel and Mihkel Kaljurand
https://doi.org/10.1016/j.cogsc.2021.100536
2452-2236/© 2021 Elsevier B.V. This is an open access article under
the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords
Gas chromatography, Liquid chromatography, Green analytical chem-
istry, Miniaturized instruments.
Introduction
Nowadays, most used chromatographic techniques
include gas chromatography (GC) and high-
performance liquid chromatography (HPLC), both
combined with different detectors, being the most
successful mass spectrometry (MS) [1].
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Green and Sustainable Chemistry
Chromatographic techniques have not been unaware of
the introduction of green practices in analytical
chemistry to ensure more sustainable and environmen-
tally friendly analysis.
GC is indeed considered a relatively green alternative
because it involves the use of inert gases as mobile
phases [2]. Nevertheless, the current impact of the
green analytical chemistry (GAC) has led to the intro-
duction of challenging changes to develop more sus-
tainable GC systems and the requirements of milder
experimental conditions, mainly involving designs shif-
ted to miniaturization [2,3].
The capacity to generate fast and precise results of
HPLC has been overshadowed from a GAC point of
view because of usage of the large amounts of solvents,
sustainability point of view include the utilization of
more sustainable mobile phases and additives for the
mobile phases, improved stationary phases, and minia-
turization [4,5].
This article intends to provide an overview on strategies
proposed to increase the greenness of HPLC and GC
methods in the last 2 years with trends particularly
focused on the miniaturization of the equipment com-
ponents, the use of more sustainable mobile phases, the
use of novel stationary phases, and the development of
novel working modes to reduce the environmental
impact of the chromatographic separations.
Figure 1 shows a summary of main improvements carried
out within the chromatographic systems, while Table 1
includes representative studies, intending to provide a
generic overview [6e23].
Advances toward green gas
chromatography
Efforts related to sustainability in GC have focused
mainly on the miniaturization of specific parts of the
equipment (mostly by preparation of microcolumns and
microdetectors) while improving the available commer-
cially portable GCs. Indeed, the field has been particu-
larly benefited by parallel advances in certain processes
Current Opinion in Green and Sustainable Chemistry 2021, 32:100536
2 Green Analytical Chemistry

Abbreviations LTM-LPGC-MS low thermal mass e low-pressure gas

chromatography coupled to mass
[P6,6,6,14][NTf2] trihexyltetradecylphosphonium
spectrometry
bis(trifluoromethylsulfonyl)imide
MEMS microelectromechanical system
ACN acetonitrile
MeOH methanol
Brij-35 nonionic surfactant
MLC micellar liquid chromatography
polyoxyethylene(23) lauryl ether
MLC-DAD micellar liquid chromatography coupled to
chip-HPLC high-performance liquid
diode array detector
chromatography on chip
MLC-PDA micellar liquid chromatography coupled to
configuration
photodiode array detector
Chip-HPLC-IMS high-performance liquid
MLC-UV micellar liquid chromatography coupled to
chromatography coupled to ion
ultraviolet detector
mobility spectrometry on chip
MS mass spectrometry
configuration
NADESs natural deep eutectic solvents
CLC capillary liquid chromatography
NEMI National Environmental Methods Index
CLC-UV capillary liquid chromatography
NEMS nanoelectromechanical system
coupled to ultraviolet detector
NP-droplet microfluidic normal-phase droplet microfluidic high-
CMC critical micelle concentration
chip-HPLC-FD performance liquid chromatography
CMV/NTD capillary microextraction of
coupled to fluorescence detector on chip
volatiles on needle-trap
configuration
CTAB cationic surfactant
PDMS polydimethylsiloxane
cetyltrimethylammonium bromide
PEG polyethylene glycol
DESs deep eutectic solvents
PID photoionization detector
EtOH ethanol
RP-HPLC reverse-phase high-performance liquid
FAMEs fatty acids methyl esters
chromatography
GAC Green Analytical Chemistry
SDS anionic surfactant sodium dodecyl sulfate
GAPI Green Analytical Procedure Index
SFC supercritical fluid chromatography
GC gas chromatography
SFC-DAD supercritical fluid chromatography coupled
GC-MS gas chromatography coupled to
to diode array detector
mass spectrometry
SIC sequential injection chromatography
HPLC high-performance liquid
SIC-FD sequential injection chromatography
chromatography
coupled to fluorescence detector
HPLC-CAD high-performance liquid
SIC-UV sequential injection chromatography
chromatography coupled to corona
coupled to ultraviolet-visible detector
charged aerosol detector
TCD thermal conductivity detector
HPLC-DAD high-performance liquid
Tween-20 nonionic surfactant polyoxyethylene(20)
chromatography coupled to diode
sorbitan monolaurate
array detector
HPLC-FD liquid chromatography coupled to
mGC micro gas chromatography
fluorescence detector
mGC-FID micro gas chromatography coupled to
flame ionization detector
HPLC-UV high performance liquid
chromatography coupled to
mGC
mGC micro comprehensive two-dimensional gas
chromatography
ultraviolet detector
ILs ionic liquids
mGC
mGC-NEMS micro comprehensive two-dimensional gas
chromatography coupled to
LP low-pressure
nanoelectromechanical system resonator
LTM low thermal mass

and technologies, such as additive manufacturing, also
known as 3D printing [10,24], microelectromechanical
system (MEMS) technology [25e28], and lithography
and etching techniques [6,29,30].
One of the most critical factors in successful GC sepa-
rations is the nature (and characteristics) of the GC
columns with other influencing factors with moderate
effects include the carrier gas selected and the tem-
perature (gradient) conditions. The GC column di-
mensions and designs allow distinguishing between
micro-GC (mGC) and traditional bench-top GC.
Current Opinion in Green and Sustainable Chemistry 2021, 32:100536
e15 m lengths, 25e1000 mm widths, and 10e800 mm
depths (for microchip-type columns) or 0.1e0.53 mm as
internal diameters di (for open tubular columns) [31].
From an environmental point of view, it is clear that mGC
involves a decrease in the mobile phase consumption, as
well as the analysis time, the latter in turn also reducing
the energy consumption. As a result, a wide variety of
columns have been proposed for mGC, including open
tubular formats [6,10,29], metallic-based [24,30], and
MEMS [25e28]. However, there are still several tech-
nical issues in these mGC columns, such as the need to
improve the obtained separation resolution and robust-
ness, the difficulties associated with the packing or
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coating of the columns with the stationary phases, the
proper obtaining of a certain column geometry (spirals
forming a square, looped, etc.) with the different
supporting materials proposed (polymers, metals, sili-
con, glass, etc.), the lack of a consensus on the best
column configuration for mGC (not only in terms of
adequacy to be coated with the stationary phase but also
considering the further connection with the remaining
components of the mGC), the difficult connection be-
tween the different pieces/parts of the system, and the
accurate control of temperature and/or pressure, among
others. These issues have certainly slowed down the
expected expansion for mGC [31,32].
Many different stationary phases are used nowadays in
mGC columns, such as ionic liquids (ILs) [6,26], silica
monoliths [27], graphitized carbon black [28], and
polymers (polydimethylsiloxane-based, polyethylene
glycol, and so on.) [9,10,24,29,30]. Efforts shift mostly
to improve the quality of the packing or coating of the
stationary phase in the specific mGC column rather than
to highlight their differential nature.
With respect to the miniaturization of GC detectors, at-
tempts have emphasized the need for better interfacing
with the column to ensure a high detection capacity,
while increasing this way the possible equipment porta-
bility [32]. The photoionization detector (PID) [25,28]
and the thermal conductivity detector (TCD) [28], for
instance, are two of the most common use detectors in
mGC because their technical characteristics simplify their
miniaturization and simpler connectivity. Likewise, de-
tectors based on sensors have been developed and tested,
such as the nanoelectromechanical system
Figure 1
Summary of main improvements in chromatographic systems to achieve greener methods.
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Green chromatography Napolitano-Tabares et al. 3
(NEMS) resonator, a sensor that can detect and monitor
analytes in accordance with their mass accumulation
through an included piezoresistive readout. These reso-
nators (Figure 2a) usually consist of a nanocantilever
made by a suspended silicon nitride film, being covered
by gold [9].
In addition to the abovementioned miniaturization of
the GC systems, new working modes have also been
proposed to fulfill sustainability requirements. Thus,
the micro comprehensive two-dimensional GC
(mGC
mGC) [9] and the microcirculatory GC [10]
constitute two representative examples of such modes.
mGC
mGC and microcirculatory GC facilitate the
analysis of complex samples, allowing even the resolu-
tion of isomeric compounds in relatively short times. In
this matter, micro comprehensive mGC
mGC is char-
acterized for the employment of two columns with
distinct properties (length and polarity) that permit the
separation and determination of analytes with different
natures at once. This technique combines miniatur-
ization and multidimensionality to address green as-
pects, not requiring an extra consumption of reagents,
sample volumes, and energy, despite the complexity
of the samples analyzed, while permitting a multi-
determination (ensuring the determination of a variety
of analytes in the same chromatographic run, instead
of requiring different GC analyses as a function of, for
example, the polarity of the compounds). A similar sit-
uation occurs with the microcirculatory GC, which is a
relatively new mGC mode, where two microcolumns are
serially connected forming a circulatory loop [10]. In this
configuration, the sample flows across the circulatory
system during several cycles to ensure the adequate
Current Opinion in Green and Sustainable Chemistry 2021, 32:100536
Current Opinion in Green and Sustainable Chemistry 2021, 32:100536

4 Green Analytical Chemistry

Table 1

Representative studies providing a generic overview on advances toward green GC and HPLC.

Technique Analytes Column Mobile phase Improvement Ref.

Gas chromatography
mGC-FID Alcohols, alkanes,
chloroalkanes, aromatic,
aldehydes, FAMEs
LTM-LPGC-MS FAMEs, drugs, mixture of
semi-volatile organic
compounds
CMV/NTD-portable GC-MS Alkenes, aromatic
compounds
mGC × mGC-NEMS High polar compounds,
alkanes
Micro circulatory GC-FID Structural isomers
Liquid chromatography
HPLC-DAD Drugs
HPLC-CAD Drugs
SFC-DAD Isoquinoline alkaloids
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Open tubular microfluidic
column (5 m × 160 mm
width × 160 mm depth)
coated with the ionic liquid
[P6,6,6,14][NTf2]
Capillary column
(2.6 m × 0.25 mm
i.d. × 0.25 mm film thickness)
coated with the commercial
stationary phase DB5MS-UI
Commercial MXT-5 column
(5 m × 0.1 mm i.d. × 0.4 mm
film thickness)
Microcolumn
(90 cm × 30 mm
width × 685 mm depth)
coated with PDMS and
microcolumn
(30 cm × 30 mm
width × 685 mm depth)
coated with PEG
OV-1 coated
microfabricated open
tubular columns (25 cm)
Ascentis® Express C18
column (100 mm × 4.6 mm,
2.7 mm) and Ascentis®
Express C18 guard column
(3 mm × 4.6 mm, 2.7 mm)
Xterra RP18 column
(150 mm × 3.9 mm, 5 mm)
Agilent Zorbax RX-SIL
column (150 mm × 2.1 mm,
5 mm)
Dry air Microfabrication of new [6]
types of mGC columns,
using ILs as stationary
phases
He Design of a fast GC method [7]
based on the use of LP
conditions and LTM
technology
He Use of portable GCs [8]
H2 Microfabrication of a [9]
compressive mGC × mGC,
and use of an alternative
detector
He Microfabrication of a [10]
microcirculatory GC system
EtOH:H2O (15:85) Substitution of acetonitrile [11]
as mobile phase by a
mixture of water-ethanol
Acetone:H2O (pH 2.5 Substitution of acetonitrile [12]
adjusted with formic acid) as mobile phase by a
(17:83) mixture of water–acetone
CO2:MeOH (0.25% DES, Use of mobile phases [13]
2% formic acid, and 2% formed by super critic CO2
H2O) (gradient mode) with methanol as additive.
The peak resolution was
improved by the addition of

a DES in the mobile phase

MLC-PDA Drugs Xterra RP18 column 0.1 M SDS, 12% Substitution of acetonitrile [14]
(150 mm × 3.9 mm, 5 mm) isopropanol in citric acid as mobile phase by
buffer (pH 5.5) aqueous solutions of
micelles and a small amount
of organic solvent
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MLC-DAD Adhesives C18 column
(150 mm × 4.6 mm, 5 mm)
MLC-UV Drugs Kinetix® C18 column
(150 mm × 4.6 mm, 5 mm)
Chip-HPLC-IMS Model mixture, isobaric Poroshell 120 EC-C18 chip
antidepressants column (35 mm × 90 mm
width × 40 mm depth,
2.7 mm)
NP-droplet microfluidic chip- (S)- and (R)- CHIRALPAK IB chip column
HPLC-FD trifluoroanthrylethanol (5 cm × 220 mm
width × 30 mm depth, 5 mm)
Portable CLC-UV Pharmaceutical compounds ODS RP capillary column
and parabens (100 mm × 300 mm i.d.,
5 mm)
Portable CLC-UV Pharmaceutical C18 capillary column
compounds, illicit drugs (100 mm × 150 mm i.d.,
Current Opinion in Green and Sustainable Chemistry 2021, 32:100536
1.7 mm)
Portable LC-UV Glycated hemoglobin Chip column
(40 mm × 500 mm
width × 500 mm depth, 5 mm)
packed with a nonporous
cation-exchange resin
SIC-UV Biogenic amines Chromolith® monolithic
silica-based cyanopropyl
(CN) column (30 × 4.6 mm)
and Supelco® Ascentis
Express superficially porous
silica-based octadecyl
(C18) (30 × 4.6 mm, 2.7 mm)
LC-FD Dyes Chip column (500 mm i.d.)
packed with polymer
monoliths based on
methacrylate-ester
SDS (0.1 M):NADES:glacial
acetic acid (92:4:4)
Brij-35 (0.01 M), SDS
(0.08 M), and NaH2PO4
(0.01 M)
MeOH:H2O (70:30)
n-Heptane:2-propanol
(95:5)
Phase A: H2O (0.1% formic
acid)
Phase B: ACN:H2O (0.1%
formic acid) (90:10)
(gradient mode)
Phase A: H2O:ACN (0.1%
trifluoroacetic acid) (97:3)
Phase B: H2O:ACN (0.1%
trifluoroacetic acid) (3:97)
(gradient mode)
75 mM NaCl in phosphate
buffer (pH 6.0)
3.0 mM phosphoric acid
aqueous solution (1D) and
7.0% (v/v) acetonitrile
3.0 mM phosphoric acid
aqueous (2D)
ACN:H2O (gradient mode)
Substitution of acetonitrile [15]
as mobile phase by
aqueous solutions of
micelles and a NADES, thus
avoiding any organic
solvent
Substitution of acetonitrile [16]
as mobile phase by
aqueous solutions of mixed
micelles, thus avoiding any
organic solvent
HPLC miniaturization to a [17]
chip, reduction in the
consumption of solvents,
and use of an alternative
detector
HPLC miniaturization to a [18]
chip, fabrication of a new
chip-HPLC, and reduction in
the consumption of solvents
Fabrication of a portable [19]
CLC, reduction in the
consumption of solvents,
and use of a miniaturized
detector
Fabrication of a portable [20]
CLC, reduction in the
consumption of solvents,
and use of a miniaturized
detector
Fabrication of a portable LC, [21]
creation of a LC microchip,
reduction in the
Green chromatography Napolitano-Tabares et al.
consumption of solvents,
and use of a miniaturized
detector
Column miniaturization, [22]
reduction in the
consumption of solvents,
and an alternative working
mode
Miniaturization of [23]
multidimensional LC and
fabrication of a new chip
5
6 Green Analytical Chemistry

Figure 2

Miniaturization in GC. (a) NEMS resonator sensor as detector for mGC × mGC. Figure adapted from the study by Whiting et al. [9], with permission from
The Royal Society of Chemistry. (b) Scheme of micro circulatory GC system. Figure adapted from the study by Hsieh and Kim [10], with permission from
Elsevier. (c) Comparison of chromatograms for the separation of the same mix of compounds using LTM-LPGC-MS (left chromatogram) and a con-
ventional GC-MS (right chromatogram). Figure adapted from the study by Fialkov et al. [7], with permission from Elsevier. GC–MS, gas chromatog-
raphy–mass spectrometry; LTM-LPGC-MS, low thermal mass – low-pressure gas chromatography coupled to mass spectrometry.

separation of the analytes. A scheme of a microcirculatory that represents its use for on-site and quick analysis,
GC system is shown in Figure 2b. these types of portable equipment keep showing soft-
ware and resolution restrictions. This, in most cases,
The adjustments on temperature programs (normally implies some difficulties in the identification of com-
implying an increase of the temperature ramps) are pounds, that is, when intending multi-analyte deter-
usually applied to achieve faster GC separations [33,34]. mination considering the column dimensions [8].
This decreases the analysis times but, at the same time,
such an increase in heating involves higher energy con-
Advances toward green liquid
sumption, which is against the GAC principles. Newer
chromatography
alternatives for faster analysis within an environmental-
The use of large amounts of solvents in HPLC (clearly
friendlier GC practice include the use of low-pressure
accompanied by a significant generation of solvent
(LP) conditions and low thermal mass (LTM) devices.
wastes) is still one of its backbones with respect to
These advances are shown in Figure 2c, together with a
sustainability. Thus, efforts to improve greenness in
comparison of chromatographic performance between
HPLC have mainly focused to decrease such
an LTM-LPGC-MS and a conventional GC-MS, by
consumption.
Fialkov et al. [7]. Low-pressure GC allows a reduction of
the analysis times together with the temperatures
To achieve such goals, novel mobile phases such as
required by the application of vacuum conditions to the
ethanol-water or acetoneewater mixtures, characterized
analytical column. In turn, this enables the system to be
by their low toxicity, good miscibility, and high biode-
coupled with a MS as detector, counteracting the asso-
gradability, have been proposed in reverse-phase high-
ciated loss of separation resolution [35,36]. On the other
performance liquid chromatography (RP-HPLC)
hand, LTM technology has led to the design of efficient
[11,12,39e44]. As disadvantage of this type of mobile
temperature programs that characterize by fast heating
phases, the ethanol-water mixture presents a high vis-
followed by fast cooling of the capillary column, without
cosity, which is accompanied by high back-pressure in
requiring additional energy consumption. As a result,
the chromatographic system. Another green mobile
this type of resistive heating device has also represented
phase proposed is pure water, used at ambient temper-
an essential advance for the commercial development of
ature, and without additives [45], but this approach
portable GCs. This can be seen in the TorionÒ T-9
clearly requires specific stationary phases (polar-
portable GC-MS from PerkineElmer or Torion Tech-
embedded).
nologies [8,37,38]. Nonetheless, despite the benefit
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 Green chromatography Napolitano-Tabares et al. 7

Figure 3

Miniaturization in LC. (a) Chromatogram of melamine using the same HPLC column and the following mobile phases: (a) 0.1 M SDS and 4% glacial
acetic acid, (b) 0.1 M SDS, 4% NADES, and 4% glacial acetic acid, (c) 0.10 M SDS, 4% glacial acetic acid, and 4% choline chloride, and (d) 0.10 M
SDS, 4% glacial acetic acid, and 4% ethylene glycol. Figure adapted from the study by Ramezani et al. [15], with permissions from Elsevier. (b)
Photograph of a capillary column adapted from the study by Albekairi et al. [63], with permission from Springer. (c) Schematic representation and
photography of a HPLC chip placed in front of the ion mobility spectrometry with shifted potentials, adapted from the study by Piendl et al. [17], with
permission from the American Chemical Society. HPLC, high-performance liquid chromatography; NADES, natural deep eutectic solvents; SDS,
sodium dodecyl sulfate.

Micellar liquid chromatography (MLC) is another well-
known greener HPLC approach used in analytical sep-
aration science since 1980 and characterized by the low
requirements of organic solvents in the mobile phase
(particularly when compared to conventional RP-
HPLC) [46e48]. MLC bases on the use of amphi-
philic additives in the mobile phase at concentrations
above their critical micelle concentration (CMC), this
way ensuring the formation of a pseudo-stationary
phase. It is important mentioning that MLC permits
the direct injection of complex samples because the
micelles of the mobile phase solubilize even proteins
and other components of the sample, while the mono-
mers of the surfactants coating the analytical columns
also help in avoiding clogging. This direct compatibility
between the samples and the mobile phase of the
chromatographic system clearly avoids analytical sample
preparation steps in many applications, which is an
additional and significant advantage of this chromato-
graphic mode in terms of sustainability. In fact, MLC
has been particularly advantageous for the analysis of
physiological samples [48e51]. Despite the low re-
quirements of organic solvents in MLC to ensure the
preservation of micelles, recent studies propose the use
of alternatives to them, such as deep eutectic solvents
(DESs) (completely avoiding the need for organic sol-
vents in the separation) [15,51]. DESs have a high sol-
ubilization capacity for polar and non-polar compounds,
thus improving the resolution and the efficiency in
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MLC (Figure 3a). Besides, in most of the cases, DESs do
not generate interferences in the UVeVis detection
region. It is important to notice that not all the DES
can be used as an additive in aqueous phases, as some
of them may decompose in presence of water into their
components [51]. Another greener approach in
MLC is based on the use of mixed micellar systems
[16,52e54]. Mixed micellar systems in MLC are
generally formed by nonionic and ionic surfactants
forming hybrid micelles that present different proper-
ties to those of the individual surfactants. The main
mixed micellar system used is the nonionic surfactant
Brij-35 þ the anionic surfactant SDS, with SDS
generating a negative charge density on the stationary
phase and Brij-35 increasing the polarity [16,52e54].
Thus, the retention of low or intermediate polar com-
pounds is reduced because of the combination of
electrostatic repulsion and hydrophobic interactions.
The strategies using mixed micellar systems in MLC
have been compared to conventional RP-HPLC sepa-
rations, to support significant improvements in terms
of chromatographic sustainability [16,52,54]. As an
example, Ibrahim et al. [16] were able to separate ten
hypertensive drugs without any organic solvent in less
than 16 min using a mixed micellar system, while in
conventional RP-HPLC it is necessary to use large
amounts of acetonitrile. The proposed chromato-
graphic method showed excellent resolution and ac-
curacy. Besides, no interferences were found when
Current Opinion in Green and Sustainable Chemistry 2021, 32:100536
8 Green Analytical Chemistry
analyzing real samples of tablet dosage forms proving
the high applicability of the method.
Another green option to avoid organic solvents is
the use of supercritical mobile phases such as CO2
[13,55e58] or H2O [59]. It is well-known that su-
percritical mobile phases have properties intermediate
between gas and liquids, such as low density, low vis-
cosity, and high diffusivity, all of them aiding to
improve the efficiency and speed in HPLC, as the
mass transfer between the stationary phase and the
mobile phase is faster. As disadvantages of supercriti-
cal fluid chromatography (SFC), pure CO2 has a low
eluotropic strength thus requiring low amounts of
methanol to succeed in the chromatographic separa-
tion. In this sense, DESs have been also explored to
improve the peak resolution and shape instead of using
organic solvents as modifiers. This effect can be
associated to the modification of the free silanol
groups over the silica surface with the DES reducing
the interactions between the analytes and the sta-
tionary phase [13]. One of the drawbacks of SFC is
that the instrumental requirements to obtain and
maintain supercritical fluids are not always affordable
in laboratories worldwide [13,55e58].
Miniaturization in HPLC has been accompanied by an
enhancement of the efficiency and detection sensitivity
[2,60,61], apart from green advantages. Among minia-
turization in HPLC in recent years, capillary GC,
Figure 4
Greenness assessment: (a) Diagrams showing the percentage of research studies on improvements in HPLC and GC methods reported between 2018
and 2020 including (or not) a green assessment in their studies. (b) Green assessment comparison between a conventional HPLC-UV method and a
MLC-UV method, both used for the analytical determination of the same group of drugs. Figure adapted from the study by Mohamed and Fouad [14]
with permission from Elsevier.
Current Opinion in Green and Sustainable Chemistry 2021, 32:100536
sequential injection chromatography (SIC), and ad-
vances due to 3D printing will be mostly addressed
given the number of studies in the recent literature.
Capillary LC is a chromatographic working mode that
employs narrow-bore columns (i.d. < 0.5 mm) that
allows increasing the separation efficiency while
consuming low amounts of solvents (as the working flow
rate needed is lower than 10 mL min1) (Figure 3b).
Besides, these kinds of columns are easily coupled to
MS detectors as the low solvent flow enhance the
ionization efficiency. However, their implementation
also requires the miniaturization of the ionization sys-
tems into chips, or to reduce the flow cell volume to few
nL in case of coupling to a UV detector [62,63].
Sequential injection chromatography (SIC) is another
miniaturized approach that combines HPLC with in-
line sample treatment and pre-concentration. SIC re-
quires a syringe pump, selection valves, and short porous
columns (2e5 cm length). The sample is injected and
submitted to a programable mobile phase flow. At the
begging, mobile phases with low elution strengths are
used to accumulate the analytes at the head of the
column. Then, the retained analytes are eluted with a
suitable mobile phase avoiding the sample pretreatment
[22,64,65]. Advantages from a GAC point of view
include the reduction of solvent consumption, the rapid
analysis, and the low cost of the instrumentation. As an
example, the consumption of organic solvents can be
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reduced from 25 mL to 0.15 mL for the determination of
salicylic acid in water samples, requiring only 8.5 min by
a SIC equipped with a fluorescence detector (FD) [65].
However, SIC is limited by the mobile phase gradient
used, making the separation of complex analytes mix-
tures difficult. In this sense, Acevedo et al. [22]have
proposed a multidimensional approach using two chro-
matographic columns to separate mixtures of biogenic
amines with different polarity. The approach permitted
the separation of the analytes in less than 8 min and
using 175 mL of acetonitrile per analysis. In comparison,
a conventional HPLC method would require two C30
columns and 30 mL of acetonitrile per run [22].
As it happens with mGC and portable GCs, current ad-
vances in 3D printing and other microfabrication pro-
cedures (photolithography, etching, and bonding
techniques) have promoted an increase in the design
and development of new chip-HPLC configurations
[17,18,22,23,66e69], detectors [17e19,66,69], and
portable HPLCs [19,20,66]. These systems have pro-
vided a wide path of possibilities to the separation field.
However, the miniaturization of HPLC into chips still
presents some challenges with respect to the detection
system, being necessary to combine them with mass
spectrometers to ensure proper sensitivity [70]. In this
sense, Piendl et al. [17]have coupled successfully for the
first time a chip-HPLC with a compact high-resolution
ion mobility spectrometer, while reducing the costs of
the detection system and the complexity as it works
under ambient pressure conditions (Figure 3c). Thus,
this has a high potential for the development of a
portable system for field analysis [17]. Indeed, the
commercial availability of these instrumental devices
has started, thus guaranteeing the expansion of their use
[19,71,72].
Green assessments in chromatographic
methods
Because GAC was first proposed, different metrics and
scales have been developed for the evaluation of
analytical methods, as a tool to determine their truthful
greenness. Among them, the eco-scale (score ranging
from 0 to 100, being 100 the best score) [73], the Green
Analytical Procedure Index (GAPI) [74], and the Na-
tional Environmental Methods Index (NEMI) [75], are
the most commonly used, as their application is user-
friendly, and the results are easy to interpret. Figure 4
shows a summary of the green assessments carried out
in recent GC and HPLC articles involving sustainability
advances. The figure also includes examples of metrics
applied.
It is important to note that the application of these
green assessment tools in GC is not extended. To the
www.sciencedirect.com
Green chromatography Napolitano-Tabares et al. 9
best of our knowledge, only one article focused on the
development of a greener GC methodology has applied a
green metric [38]. In such a study, the greenness and
analytical performance of a portable GC were compared
with that of a conventional bench-top GC. The results
of the application of the eco-scale indicated that the
portable GC increased the greenness of the method,
mostly for the advantages of on-site measurement.
Nevertheless, the green assessment paid particular
focus to the sample preparation technique used and not
to the chromatographic method.
On the other hand, the use of green metrics in HPLC
studies is more extended than for GC, but it is not
either a universal practice. In percentage, up to 30% of
the HPLC studies included such evaluation, as shown in
Figure 4. Interestingly, the metrics have been mostly
used to assess the sustainability of alternative solvents
in HPLC and not to evaluate the greenness associated
with the miniaturization of the equipment components
[11,12,14,16,39e41,44,48,52,54,76,77]. With respect to
HPLC studies on the substitution of organic solvents,
the eco-scale results show a clear improvement in most
of the cases, with an overall score of 90 points. In any
case, as it happened with GC studies, the evaluation of
the impact exerted by the miniaturization on the
method greenness is a pending topic. Only one article
that follows a SIC strategy has evaluated the impact of
miniaturization [64]. In such a study, the elimination of
previous sample preparation steps together with the
miniaturization of the column size considerably
increased the sustainability of the proposed approach
when compared with conventional HPLC systems.
Indeed, as observed in the punctuation of the method, it
got a score of 91 for SIC, whereas the conventional
HPLC method scored 55.
Conclusions
The advances toward greener chromatographic methods
have been mainly focused on miniaturization, thanks to
the advantages achieved last year in 3D printing,
MEMS, lithography, and etching techniques. Miniatur-
ization clearly involves reduction of mobile phases
consumption and, therefore, in wastes generation, as
well as decreases in energy requirements. Such minia-
turization has also benefited by advances in novel sta-
tionary phases to prepare novel microcolumns. Among
miniaturized approaches in chromatography, those
added by multidimensional methods also include the
possibility of solving complex mixtures within sustain-
ability. Thus, mGC, mGC
mGC, chip-GC, and portable
GC, should be highlighted with respect to greener
miniaturized GC. Chip-based HPLC, SIC, and portable
HPLC should be highlighted with respect to greener
miniaturized HPLC.
Current Opinion in Green and Sustainable Chemistry 2021, 32:100536
10 Green Analytical Chemistry
Apart from miniaturization and novel stationary phases,
HPLC also involved other green improvements, with
the use of greener mobiles phases (in MLC and SFC,
mostly), while testing DESs both as main mobile phases
and additives to improve sustainability.
In any case, it is important to highlight that all the
modifications carried out in HPLC and GC not only
involved an increase in the sustainability of the
technique but also improvements in the chromato-
graphic resolution and sensitivity of the resulting
methods.
Last but not least, it is important to consider that
claimed greenness in these approaches should be
supported by proper green metrics. Few studies (almost
none in GC) have covered this type of evaluation, and in
general, none for the miniaturization of HPLC and GC.
It is also true that such scales should require certain
improvements to truly give a vision of the improvements
achieved toward green chromatography, and pending
efforts for these metrics are still required.
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.I.N.-T. and A.G.-S. thank the Agencia Canaria de Investigación, Innova-
ción y Sociedad de la Información (ACIISI), co-funded by the European
Social Fund, for their FPI PhD fellowships. V.P. thanks the Spanish Min-
istry of Economy, Industry and Competitiveness (MINECO) for funding
the research proposal with grant number MAT2017-89207-R and the
ACIISI for the research proposal with grant number ProID2020010089.
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