Journal of Chromatography A 1643 (2021) 462034

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Automated method for volatile fatty acids determination in anaerobic

processes using in-syringe magnetic stirring assisted dispersive
liquid-liquid microextraction and gas chromatography with flame
ionization detector
M.A. Vargas-Muñoz a, Víctor Cerdà a,b, L.S. Cadavid-Rodríguez c, Edwin Palacio a,∗
a
Group of Environmental Analytical Chemistry, University of the Balearic Islands, Cra.Valldemossa km 7.5, 07122 Palma de Mallorca, Spain
b
Sciware Systems, S.L., C/Pi 37, 07193 Bunyola, Spain
c
Department of Engineering, Faculty of Engineering and Administration, Universidad Nacional de Colombia - Sede Palmira, Cra. 32 No 12-00, Palmira,
Colombia

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

Article history: Volatile fatty acids (VFAs) are key parameters to monitor anaerobic digestion processes. Thus, a fast, sim-
Received 12 December 2020 ple and precise determination of these analytes is necessary for a timely characterization of the biological
Revised 19 February 2021
processes present in municipal solid waste and wastewater treatment plants. In this work, an automated
Accepted 28 February 2021
method for the extraction and preconcentration of VFAs, based on dispersive liquid-liquid microextraction
Available online 10 March 2021
with magnetic stirring in syringe, and gas chromatography with flame ionization detector for the sepa-
Keywords: ration and detection, is described. The effect of parameters such as the type and volume of extraction
In-syringe magnetic-stirring-assisted solvent, pH, salting out effect and stirring time, was studied using a multivariate and univariate experi-
dispersive liquid-liquid microextraction (In mental design. Extraction and preconcentration were performed simultaneously using tert-butyl methyl
syringe-MSA-DLLME) ether (TBME) as the extraction solvent, after stirring 100 s at a constant rate. The detection limits were in
Gas chromatography the range of 0.1 - 1.3 mg L−1 and a good linearity was observed up to 10 0 0 mg L−1 of the studied VFAs,
Volatile fatty acids
with a range of R2 between 0.9997 and 0.9999. The intra and interday precision expressed as relative
Anaerobic treatment, automation
standard deviation (n= 5) varied between 0.7 and 2.4% and between 1.7 and 7.0%, respectively. Subse-
quently, the developed method was successfully applied to evaluate the presence of VFAs in wastewater
samples from anaerobic treatments and an average relative recovery of 102% was obtained.
© 2021 Elsevier B.V. All rights reserved.

1. Introduction tors present in wastewater treatment plants (WWTP), solid waste

Volatile fatty acids are aliphatic carboxylic acids with low
molecular weight and short chain lengths (C2-C7) [1]. They are in-
termediate metabolites originated by microbial conversion of or-
ganic matter into biogas during anaerobic digestion (AD) and are
mainly formed from the fermentation of macromolecules (carbo-
hydrates, proteins and fats) in acidogenesis and acetogenesis steps
[2]. The main components of VFAs present in anaerobic digesters
are acetic, propionic, butyric, valeric acid and the isomeric forms
of these last two [3,4].
AD is a process used as a biological treatment of wastewater,
sewage sludge and organic solid waste with the aim of reducing
environmental pollution and producing renewable energy in the
form of biogas [5]. VFAs are a key indicator in anaerobic reac-
∗
Corresponding author.
E-mail address: edwin.palacio@uib.es (E. Palacio).
treatment plants (SWTP) and in landfill leachates [6], because large
amounts of VFAs (>10 0 0 - 40 0 0 mg L−1 ) can inhibit the AD pro-
cess [7], affecting the stability of methane production and the re-
moval of nutrients (nitrogen and phosphorus compounds) from
wastewater [8]. Otherwise, a high concentration of VFAs is bene-
ficial to generate value-added products with different uses in the
chemical industry [9,10].
VFAs should be monitored at different stages of wastewater
treatment, as well as in the untreated wastewater, treated wastew-
ater and activated sludges [11]. There are traditional procedures
based on titration, which only determine the total concentration
of volatile fatty acids (TVFAs). However, the determination of in-
dividual VFAs gives more specific information about the biolog-
ical processes; thus, their separation has become more impor-
tant over time. Individual VFAs can be separated with analytical
techniques such as gas chromatography (GC), high performance
liquid chromatography (HPLC), ion chromatography and capillary
electrophoresis. Of these techniques, GC with flame ionization de-

https://doi.org/10.1016/j.chroma.2021.462034
0021-9673/© 2021 Elsevier B.V. All rights reserved.
M.A. Vargas-Muñoz, V. Cerdà, L.S. Cadavid-Rodríguez et al.
tection (GC-FID) is the most used for the VFAs determination in
wastewater because it provides a high resolution, wide linearity
and low limits of detection [12].
Recently, the beginning of using polar columns (FFAP) in GC
motivated the possibility of performing a direct aqueous injection
(DAI) of the samples to avoid the loss of analyte during enrich-
ment step [7,13]. However, numerous studies states that DAI can
be detrimental to the analytical performance of the GC, observ-
ing wide peaks, column contamination and detector damage. For
this reason, sample preparation is essential to protect the instru-
ment [12,14] using treatment techniques such as liquid-liquid ex-
traction (LLE) [11,15], solid phase microextraction (SPME) [16,17]
and headspace (HS) techniques [18]. Among these treatment tech-
niques, LLE seems to be the most favorable option because it is ac-
cepted as a practical, fast and suitable technique for a large num-
ber of samples. LLE allows analyte preconcentration and sample
matrix elimination. However, LLE requires extended sample prepa-
ration times, large volumes of solvents and therefore, a costly dis-
posal thereof [19].
In this context, unconventional LLE methodologies have
emerged, gathered in the techniques of liquid phase microextrac-
tion (LPME); miniaturized LLE procedure consisting of the ex-
traction of objective compounds from an aqueous matrix using
reduced volumes of an organic immiscible solvent [20]. LPME
techniques include the dispersive liquid-liquid microextraction
(DLLME), a technique based on the use of a dispersive solvent,
which has the function of dispersing the organic phase in very fine
droplets in the aqueous phase, increasing the contact surface be-
tween the two phases and, therefore, improving the extraction effi-
ciency [21]. Nonetheless, the use of dispersive solvents can change
the partition coefficient and decrease of the mass transfer of the
analytes to the extraction solvent, reducing the enrichment effi-
ciency [22]. To overcome this problem, alternative methods to dis-
perse the extraction solvent have been implemented to replace the
dispersion solvent using different methods of mechanical agitation:
ultrasonic-assisted [23], air-assisted [24], vortex-assisted [25] and
magnetic stirring-assisted [26]. Among these, magnetic stirring-
assisted dispersive liquid-liquid microextraction (MSA-DLLME) is
based on the dispersion of an organic solvent in the aqueous sam-
ple using magnetic stirring to form a mild emulsification [26].
Nevertheless, the manual methodology of a DLLME can become
slow, laborious and imprecise. In this sense, flow analysis tech-
niques and automation are a viable option, because they provide
high performance, low analyst intervention, lower solvent con-
sumption, and therefore a low hazardous waste generation for en-
vironment [27]. The use of automated chromatographic systems in
large-scale AD plants [13,28,29] and in laboratory [30,31] has al-
ready been reported. Thus, MSA-DLLME can be automated using
the syringe laboratory technique, allowing improved reproducibil-
ity and precision, giving place to In-syringe-MSA-DLLME [32].
In the present work, the automation of a dispersive liquid-
liquid microextraction method with magnetic stirring in syringe
(In-syringe-MSA-DLLME) was developed for the VFAs determina-
tion in an anaerobic digester of the WWTP from Palma of Mallorca
(Spain) and in laboratory scale biodigesters for fishing waste val-
orisation. The configuration of the analytical system is based on
the multi-syringe flow injection analysis (MSFIA) technique, but a
single syringe is used as the extraction chamber. A magnetic stir
bar is placed inside the syringe and its rotation is controlled with
a motor that is activated by a computer. In this way, the agitation
disperses the extraction solvent within the aqueous sample. Then,
the organic phase is separated from the aqueous phase, and the
organic drop enriched with the analytes is transported through the
manifold and stored until its analysis by GC-FID. Several extraction
solvents with different densities have been studied, in addition to
the optimal conditions of pH, salinity, extraction time and extrac-
2
Journal of Chromatography A 1643 (2021) 462034
tant volume. Finally, a comparison of the proposed method is made
with other conventional methods, focusing mainly on the distilla-
tion method for VFAs [33].
2. Materials and methods
2.1. Reagents
Individual C2-C5 VFAs standards (≥99%) and 2–ethylbutyric
acid (≥99%) used as internal standard (IS) were obtained from
Sigma-Aldrich (Madrid, Spain). Certified VFAs mix at a concen-
tration of 10 mM was obtained from Supelco (USA). The extrac-
tant solvents tert-butyl methyl ether – TBME (HPLC grade) (≥99%)
and dichloromethane were also obtained from Scharlau (Barcelona,
Spain), as well as phosphoric acid (85% w/w). The dimethyl car-
bonate extractant was purchased from Sigma Aldrich. Sodium chlo-
ride was obtained from Scharlau. The distilled water came from the
MiliQ Direct-8 purification system (Millipore Ibérica S.A.U., Madrid,
Spain).
2.2. Standards
From the individual high purity VFAs standards (≥99%) a stock
solution was made at 2500 mg L−1 in distilled water. The stan-
dards for direct injection were prepared by dilution in TBME.
Working solutions (5 – 10 0 0 mg L−1 ) were prepared from the stock
solution and 2-ethylbutyric acid was used as an internal standard
(IS) with a final concentration of 250 mg L−1 . Sodium chloride
and phosphoric acid were used to adjust salt content and pH of
aqueous solutions in optimization studies, respectively. The inter-
nal standard chosen and the concentration of acidifying solution
was prepared according to recommendation of Raposo et al, 2015
[1]. Calibration was done by using relative peak area (RPA=VFA
peak area / IS peak area).
2.3. Samples collection
Liquid samples were obtained from wastewater from the inlet
and outlet of the WWTP (Palma de Mallorca, Spain) and from an
anaerobic digester treating sludge from the primary and secondary
treatment of wastewater. Further, samples from three laboratory-
scale fermenter reactors (FR) (located at the Universidad Nacional
de Colombia – Sede Palmira) were analyzed. The reactors were
designed to ferment 50 g SV L−1 of fishing waste (FW) at three
pH values (7, 9 and 11) with inoculum-substrate ratio of 0.5. The
FW were the viscera of different species of fish (mainly snappers,
corvines and tunas) obtained from artisanal fishing in Tumaco,
Colombia. The samples were taken on days 1, 3, 5, 7, 10 and 15
of AD. The samples from the WWTP and FR were stored in the
dark at -20°C in 1 L and 30 mL polypropylene bottles, respectively.
Subsequently, they were acidified with phosphoric acid (85% w/w)
to ensure the predominance of undissociated forms of VFA species.
Once in the laboratory they were passed through a 0.45 μm filter
to remove solid particles.
2.4. In-syringe-MSA-DLLME-GC-FID system
The main element for the treatment system (Fig. 1) is a multi-
syringe burette module (Crison, Alella, Spain) coupled to a multi-
position valve (MPV). Multisyringe module (MSM) can be equipped
with four syringes that are moved simultaneously in two directions
to aspirate or dispense liquids. In the proposed system it was only
necessary to use a 5 mL glass syringe (S) (Hamilton, Switzerland),
which also acts as a mixing chamber. The syringe has a three-way
solenoid valve on its head, performs the multicommutation opera-
tion.
M.A. Vargas-Muñoz, V. Cerdà, L.S. Cadavid-Rodríguez et al.
Fig. 1. Automatic In-syringe-MSA-DLLME-GC-FID system for VFAs extraction and preconcentration. V, three-way solenoid valve of syringe; S, syringe; MSM, multisyringe
module; MPV, multiposition switching valve; SV, solenoid valve; AS, autosampler, M, motor.
The on position of the syringe is connected with the central
port of the MPV and the off position joins to an additional exter-
nal solenoid valve (SV). On the back, the multisyringe module has a
panel with four ports which allows controlling and connecting the
motor and the SV (Fig. S1A). So, these devices are first connected
to an electronic circuit to prevent overheating (Fig. S1B). The mul-
tiposition valve (MPV) (Sciware Systems, Bunyola, Spain) has eight
peripheral ports, as follows: extractant solvent container (6), sam-
ple (5) and air (4). The sample tube connection extends to an au-
tosampler system, which is equipped with chromatographic vials
containing 300 μL inserts. The MSA consists of an external stirring
device (Sciware Systems, Bunyola, Spain) that rotates a small stir
magnetic bar (10 mm long and 3 mm in diameter) inside the sy-
ringe; a motor that causes the rotational force of the external stir-
ring support, connected with an elastic band (Fig. S1C); and a cir-
cuit, which controls the motor speed through one of the rear out-
puts of the multisyringe module (Fig. S1D). The MSA is described
in detail in the authors work [26].
Both modules were connected to a computer, and instrumen-
tal control is done through AutoAnalysis 5.0 software (Sciware Sys-
tems). All manifold networks used in the systems have been man-
ufactured with 0.8 mm internal diameter PTFE tube, except that
used for solvent inlet in the selection valve (1.5 mm). The screws
and connectors are made of PEEK, so that they are resistant to or-
ganic solvents.
2.5. GC-FID instrumentation
The analysis was performed on a YL-Clarity YL6500 gas chro-
matograph, equipped with an automatic sample injector and a
flame ionization detector (FID) of the same brand. The analytes
were separated on a polar capillary column of fused silica with
polyethyleneglycol (DB-FFAP, Agilent, 30 m x 0.25 m id x 0.25 μm).
3
Journal of Chromatography A 1643 (2021) 462034
Nitrogen (99.99%) was used as carrier gas at 1 mL min−1 . The oven
temperature was planned to increase from 100°C (2 min) to 150°C
(1 min) to 10°C min−1 and then rose to 200°C (1 min) to 20°C
min−1 , for a time 11.5 min total analysis. The injection volume of
the sample was 1 μL and was performed in 50:1 split mode at
250°C. The temperature of the detector was maintained at 250°C.
2.6. In-syringe-MSA-DLLME-GC-FID procedure
The detailed operation protocol of the proposed method is
showed in supporting information (Table S1). Prior analysis, salt
content and pH of samples were adjusted to 0.5 M using NaCl
and pH 1.0, respectively. The cleaning tubes, sample tubes, waste
tubes and chromatography vials were strategically located in the
autosampler, thus the liquid that enters through a specific sample
or cleaning tube goes out to 13 positions later in a vial or in the
waste tubes, respectively (Fig. S1E). The speed used in the motor
was always constant.
2.7. Calculations
Enrichment factor and extraction recovery (ER) were studied in
order to evaluate the extraction efficiency. The enrichment factor
(EF) was calculated as the ratio of Corg /C0 , where Corg is the con-
centration of analytes in the organic phase after DLLME and C0 the
original concentration in the aqueous phase. The ER was defined
as the percentage of the total analyte which was extracted in the
C x Vorg
organic drop after DLLME (ER = org C x Vaq x 100 ) . Relative recovery
0
(RR) was calculated as the percentage of the found concentration
after spiking, minus real concentration in the samples before spik-
ing, relative to the added (spiked) concentration in the samples
Cfound− C
(RR = Cspiked
real
x 100).
M.A. Vargas-Muñoz, V. Cerdà, L.S. Cadavid-Rodríguez et al.
Fig. 2. (A) Results of the study of extractants with error bars. Results are expressed as relative intensity (%) giving the 100% to the solvent showing the best performance
and relating the rest to it. DMC: dimethyl carbonate, TBME: tert-butyl methyl ether, DCM: dichloromethane. (B) Standardized main Pareto chart from the fractional factorial
screening design for the calculated desirability response signal. Vertical line in the chart defines 95% of confidence level. (C) Effect of stirring time on the response of the
analytical signal (area) of the TVFAs. (D) Results of the study of salting out effect with error bars. Results are expressed as relative intensity (%) giving the 100% to the salt
concentration showing the best performance and relating the rest to it.
Linearity, sensitivity, quantification, precision and accuracy were
calculated according to the IUPAC [34]. LOD and LOQ were calcu-
lated, as three times the standard deviation of ten blanks divided
by the slope of each calibration curve, and ten times the standard
deviation of ten blanks divided by the slope of each calibration
curve, respectively. Precision and accuracy was evaluated through
recovery studies [35].
2.8. Statistical analysis
Design of experiments and results evaluation were carried out
using the statistical software package “STATISTICA 6.0” (Statsoft
Inc., Tulsa, USA). All calibration solutions and samples were an-
alyzed in triplicate. The comparison of the proposed automated
method with the 5560C distillation method was carried out with
XLSTAT 2019.1 (Addinsoft SARL., Paris, France) statistical program.
3. Results and discussion
3.1. In-syringe-MSA-DLLME
3.1.1. System cleaning
The rinsing of system is necessary to minimize carry-over of
analytes between extractions. Adsorption of VFAs can occur mainly
in the port number 5 of the selection valve, inside the syringe
and in the PTFE tubing. Two, three and four washes were tested
(n= 2) in the syringe after treating the most concentrated stan-
dard (10 0 0 mg L−1 ). "Two washes" are composed of a cleaning
performed with 2 mL of acetone (10% v/v) and then, with 2 mL
of water. "Three washes" are composed of a cleaning with acetone
and then twice with water, in the same volumes. "Four washes" in-
clude two cleanings with water and two cleanings with acetone. It
4
Journal of Chromatography A 1643 (2021) 462034
was found that "three washes" were necessary to perform a good
cleaning of the syringe (not shown in this work). There was no sig-
nificant minimum difference between three and four washes (p=
-0.033). Therefore, the procedure was worked with three washes
and took an approximate time of 4 min.
3.1.2. Selection of extractant solvent
A critical step in the development of an extraction method
is the selection of an appropriate solvent to extract the ana-
lytes from the sample. The optimization of the extraction condi-
tions was studied for acetic, propionic, isobutyric, butyric, isova-
leric and valeric acids. A good solvent should have a high analyte
extraction capacity and a good chromatographic behavior. Solvent
selection was carried out using tert-butyl methyl ether (TBME),
dichloromethane (DCM) and dimethyl carbonate (DMC), to evalu-
ate the VFAs extraction efficiency. These solvents were chosen be-
cause they are the most used and named in recent studies to ex-
tract VFAs [7,11,15]. It should be noted that TBME is a less dense
solvent than water, unlike DCM and DMC. The use of alcohols was
avoided due to their high miscibility in water. For this test, a 3
mL aqueous sample enriched with 500 mg L−1 was extracted with
500 μL of the solvent, at pH 1.0 and without salt. To achieve a good
mix and then a total phase separation, the stirring time and sepa-
ration time were set at 120 and 20 s, respectively. The experiment
was performed in triplicate. Results showed that DMC achieved the
highest extraction efficiency for all studied analytes with respect
to other solvents (Fig. 2A). However, this solvent is slightly misci-
ble in water and it was not chosen because it did not provide a
good phase separation after stirring. Therefore, TBME was selected
as the optimal extraction solvent. TBME generally has a greater re-
covery for all analytes compared to DCM, due to its higher polar-
ity [15]. Dichloromethane has a lower affinity with the more po-
M.A. Vargas-Muñoz, V. Cerdà, L.S. Cadavid-Rodríguez et al.
lar analytes (acetic and propionic acids); important acids to de-
termine the biodigestion efficiency [36]. Moreover, a solvent less
dense than water such as TBME is a more environmentally friendly
solvent compared to DCM [30].
3.1.3. Sample volume selection
The effect of sample volume on extraction was also investi-
gated. As expected, when the volume of the enriched aqueous so-
lution was increased from 1 to 3 mL, the analytical signal increased
linearly due to the greater availability of the analyte mass for ex-
traction. An average increase of 2.2 times was obtained for all an-
alytes. Volumes greater than 3 mL were not analyzed because it
was necessary to save space for the volume of the extraction sol-
vent (studied from 0.2 to 0.5 mL) and to aspirate the minimum
volume of air (1.2 mL) necessary to introduce the sample and the
solvent into the 5 mL syringe. Therefore, 3 mL was chosen for the
following experiments in view of high sensitivity.
3.1.4. Multivariate and univariate optimization of the
in-syringe-MSA-DLLME
Once the extraction solvent was selected, the optimization pa-
rameters affecting the DLLME was developed using the protocol
detailed in Table S1. The objective of optimizing the analytical
method is to find the best system conditions and is measured in
general, in terms of sensitivity of the analytical signal. This can be
done as a univariate analysis, studying a single variable while the
other factors remain constant, or as a multivariate analysis, design-
ing experiments where all the variables and their interactions are
studied at the same time. In the present study a combination of
the two methodologies was used to show greater precision of the
results. Statistical software Statistical 6.0 (Statsoft, Tulsa, USA) was
used for all multivariate optimization.
First, a preliminary study was carried out to find the variables
with a significant effect on the response, through a fractional factor
evaluation (2k-1 ), including three central points. For the screening
test, 11 experiments were performed in triplicate and four vari-
ables were covered with wide evaluation ranges: extractant vol-
ume (20 0-50 0 μL), salt content (0.0 - 1.0 M), pH (1.0 - 2.0) and
stirring time (40 - 120 s). The peak area of each VFA was used as
the response signal. The sum of normalized multiple responses of
the analyte area was used to transform the individual responses of
each acid into one. For this, the area of individuals VFAs in each
of the experiments was divided over the largest area obtained for
said analyte under the optimization conditions developed. For this
research, 3 mL of sample enriched with 500 mg L−1 was used.
The use of dispersants was evaluated, however it was not used
because previous tests with TBME solvent and dispersants such as
acetone, methanol and acetonitrile showed a dispersion of micro-
droplets that did not separate into two phases. This fact is possi-
bly attributed to the similar densities between the chosen extrac-
tant and the dispersants. However, not using an additional solvent
brings the benefit of having a lower reagent expense and avoid
dilution of the analytes in the medium. In addition, conventional
liquid-liquid extraction procedures for VFAs usually only use a sin-
gle organic solvent. Likewise, thanks to the small vortex generated
by motor it is possible to form the dispersion. The fastest speed al-
lowed by the MSA system was fixed (equivalent to 1.4 V). A rapid
separation between the organic phase and the aqueous phase was
observed and, therefore, the separation time was set at 20 s.
The results obtained in the Pareto chart (Fig. 2B) and in ANOVA
test (Table S2) showed that the curvature, extractant volume, pH,
salt concentration and extraction time were significant with a 95%
confidence level, indicating that optimum values could be found
within the studied range. As expected, the Pareto chart displays a
negative effect on the extractant volume and pH, showing that at
5
Journal of Chromatography A 1643 (2021) 462034
lower volumes of TBME and more acidic pH improves the extrac-
tion. On the other hand, salt concentration and stirring time has
a significant positive effect, so adding salt and stirring longer in-
creases the extraction. The interaction between the extractant vol-
ume and the stirring time was the most significant factor showing
a negative effect.
As can be seen in the desirability profile of the preliminary
study (Fig. S2A), the optimum values within the studied range
were: 200 μL, an extraction time of 120 s, a pH of 1.0 and a salt
concentration of 1.0 M. However, variables with significant effect
on a central composite design (CCD) were evaluated to find the
optimum values (Fig. S2B). Based on the above, it was decided to
study again the volume of the extractant between 150 and 300
μL, and the extraction time between 60 and 180 s in a two-factor
CCD with three central points (n= 3) showing that the optimum
values are a minimum volume for the extractant (120 μL) and a
maximum time for agitation (205 s). However, despite these re-
sults, there were many problems with the recovery in the vial of
such small volumes of organic extract and reproducibility problems
were presented when trying to extract with volumes smaller than
200 μL. When very low volumes of extractant are used, there is
a risk of dilution of the organic drop along the pipe, resulting in
the observation of aqueous phase microdroplets in the VFAs ex-
tract, which may change the recovered concentration and affect
the performance of the chromatograph column. Therefore, taking
into account the small difference between the ranges of the vol-
umes studied, it was decided to take 200 μL as the most appro-
priate volume.
On the other hand, stirring times up to 10 min were tested in
a univariate analysis and 100 s was chosen as the most suitable
value for extraction (Fig. 2C). Although pH and saline concentra-
tion were significant factors in the Pareto chart, it was not decided
to evaluate these parameters in the CCD and they were set as 1.0
and 0.5 M, respectively. The pH of the sample had a negative effect,
therefore, a pH 1.0 is safe to have the VFA in its molecular form,
favoring even better the undissociated form of VFA with a lower
pKa such as acetic acid (pKa= 4.75). A deeper univariate analysis
was performed for the salting out effect (Fig. 2D). Finally, a robust-
ness validation test was performed in a two-factor complete facto-
rial design (stirring time and extraction volume) with three central
points (Fig. S2C). The data obtained show a robust system toler-
ant to an oscillation of 5% over the optimal value of the variables,
working with a 95% confidence (p= 0.05).
3.1.5. Stirring time
The effect of shaking for longer times on the analytical response
has been evaluated (Fig. 2C), showing that equilibrium is reached
for all objective analytes after 7 min. It should be noted that the
extraction for acetic at 500 mg L−1 did not increase after 100 s,
but the extraction of longer chain acids than acetic acid contin-
ued to increase over time, contributing to a rapid increase in the
TVFAs concentration. In a previous experiment (not shown here)
it was observed that 100 s was adequate to perfectly observe 5
mg L−1 of acetic acid and even lower concentrations of the same
acid as was shown later in the method validation. Likewise, per-
forming extractions with long times can lead to greater miscibility
between the aqueous and organic phases, increasing the time for
phase separation. Thus, based on the previous discussion and the
results obtained with the preliminary analysis and the CCD, where
it was observed a small difference between 100 and 120 s, it was
decided to choose 100 s to perform the extraction. This, since the
sensitivity of the proposed method is in accordance with the con-
centrations present in the analyzed matrices, and at the same time
the requirements of longer stirring times are reduced.
M.A. Vargas-Muñoz, V. Cerdà, L.S. Cadavid-Rodríguez et al. Journal of Chromatography A 1643 (2021) 462034

Table 1
Figures of merit of the developed In-syringe-MSA-LLME method for VFAs determination.

Acetic Propionic Isobutyric Butyric Isovaleric Valeric

Retention time (min) 5.16 ± 0.05 6.11 ± 0,05 6.42 ± 0.05 7.12 ± 0.05 7.63 ± 0.05 8.51 ± 0.05
LOD (mg L−1 ) 1.3 1.0 0.8 0.5 0.3 0.1
LOQ (mg L−1 ) 4.3 3.3 2.5 1.8 1.1 0.2
Linear range (mg L−1 ) 4.3 - 1000 3.3 - 1000 2.5 - 1000 1.8 - 1000 1.1 - 1000 0.2 - 1000
Slope ± SD 0.0555 ± 0.002 0.2712 ± 0.003 0.5716 ± 0.002 0.5426 ±0.002 0.7681 ± 0.003 0.7966 ± 0.001
Intercept ± SD 0.0003 ± 0.00006 0.0007 ± 0.00003 0.0001± 0.00002 0.0034 ± 0.0002 0.0034 ± 0.0002 0.0039 ± 0.0006
Determination coefficient (R 2 ) 0.9998 0.9999 0.9997 0.9998 0.9999 0.9999
Intra-day precision (%) (n= 5) 1.3 2.4 2.3 2.3 1.0 0.7
Inter-day precision (%) (n= 5) 6.9 3.6 3.0 1.7 1.9 2.1
Enrichment factor 1.2 3.2 4.4 4.3 5.3 5.5
ER to 10 mg L−1 (%) (n= 5) 5.7 20.0 31.6 30.1 32.4 38.5
ER to 50 mg L−1 (%) (n= 5) 6.5 22.7 28.5 29.4 36.1 37.0
ER to 100 mg L−1 (%) (n= 5) 7.7 22.1 27.8 26.4 37.5 35.1

SD: standard deviation, ER: extraction recovery.

3.1.6. Salting out effect
The effect of salt content was evaluated, since it sometimes
produces an improvement in extraction efficiency [7,11]. Therefore,
NaCl concentrations were added up to 2.0 M. According to Fig. 2D,
the extraction efficiency after adding 0.5 M of NaCl increased for
acetic 38%, for propionic 37% and for the rest of the fatty acids an
average increase of about 20% was obtained. There were no signif-
icant differences between 0.5 M and 1.0 M, but a concentration of
2.0 M slightly decreased VFAs extraction. Therefore, a salt concen-
tration of 0.5 M was chosen. The addition of salt to a water sam-
ple increases the extraction of hydrophobic and hydrophilic ana-
lytes. This reduces hydrogen bonds between hydrophilic analytes
and water, which reduces their solubility in water and increases
their mass transfer to the organic phase. However, when the con-
centration of sodium chloride increases, the extraction efficiency of
fatty acids decreases slightly, due to an increase in viscosity caused
by the interactions between the ionic form of the salt and these
polar analytes.
3.2. Figures of merit of the in-syringe-MSA-DLLME-GC-FID method
To evaluate the performance of the proposed method, its fig-
ures of merit were studied and shown in Table 1. The linear work
range was established from the LOQ to the highest standard, show-
ing correlation coefficients (R2 ) greater than 0.9997. As can be seen
in this table, a wide range of concentrations were quantified, i.e.,
4.3-10 0 0 mg L−1 of acetic acid, 3.3-10 0 0 mg L−1 of propionic, 2.51-
10 0 0 mg L−1 of isobutyric acid, 1.8 - 10 0 0 mg L−1 of isobutyric
acid, 1.1 - 10 0 0 mg L−1 of isovaleric and 0.2 - 10 0 0 mg L−1 of va-
leric. LODs were lower than 1.3 mg L−1 and LOQs were lower than
4.3 mg L−1 . In this case, the LOD are only estimates to verify the
absence of interference during the VFAs analysis, since typical con-
centrations in real samples do not require the detection of trace
quantities. The intra-day and inter-day precision were determined
by analyzing five spiked samples from anaerobic digester of the
WWTP of Palma de Mallorca (AD) at 50 mg L−1 within same day
and during five different days, respectively. As can be seen, RSD
were found from 0.7 to 2.4% in one day and from 1.7 to 6.9% in
five days (n= 5).
Accuracy of the proposed method was evaluated in five repeti-
tions by spiking 10, 50 and 100 mg L−1 to the anaerobic digester
sample. Enrichment factors were between 1.2 to 5.5. Recovery rates
varied from 5.7 to 38.5%. An increase in extraction efficiency for
acids having a longer chain was observed. Although these values
were low, the preconcentration carried out is more than enough to
detect low concentrations of VFAs, since in this case it works with
concentrations in the range of mg L−1 , due to the characteristics
of the studied matrix, a high preconcentration of the analytes is
not required. Likewise, the most important reason to transfer the
analytes to the organic phase lies in the fact of avoiding the prob-
lems derived from the injection of water into the GC. Therefore,
the method developed has good performance and great applicabil-
ity for wastewater samples.
3.3. Application to wastewater samples
In order to evaluate the applicability of the method developed
in syringe, 4 types of wastewater samples with different matri-
ces were analyzed (Table 2). Sample 1 comes from the inlet (I) of
wastewater in the WWTP of Palma de Mallorca, sample 2 corre-
sponds to the outlet (O) of the purified water after the complete
treatment, sample 3 was obtained from anaerobic digester super-
natant (AD) which treats the residual sludge from the process of
primary and secondary treatment of the wastewater, and sample
4 comes from the laboratory-operated (FR) fermenter reactor (day
10, pH 7.0). The results showed that only sample 4 (FR) contained
appreciable amounts of all the VFAs analyzed (Fig. 3).
On the other hand, no VFAs were found in the WWTP samples
above the LOD. The possible absence or low content of VFAs in the
AD sample can be attributed to the rapid transformation of VFAs
into biogas, indicating the efficiency of the process and an absence
of inhibition in methane production [37]. Therefore, to evaluate the
effects of the matrix, all wastewater samples were spiked with the
target analytes at 10, 50 and 100 mg L−1 and recovery studies were
performed. Samples were also spiked with a certified VFAs stan-
dard at a concentration of 1 mM for method validation.
As can be seen in Table 2, a good recovery of the standards
added to the four samples of wastewater was obtained, with a
general average of 102.5 ± 8.2 %. However, the relative recovery
(RR) of the longer chain VFAs (100 mg L−1 of isovaleric and va-
leric acids) of the inlet sample was slightly lower with respect to
the RR percentages found for the other samples. This is attributed
to the wastewater matrix in the inlet is much more complex be-
cause it has not undergone any treatment as of the other sam-
ples analyzed did, and possibly a large variety of higher molecular
weight compounds (carboxylic acid of longer aliphatic chain, car-
boxylic acids having an aromatic cycle, carbohydrates, amino acids,
alcohols, others [38]) in the sampled aliquot generated some type
of interference at the analysis time. Finally, these results demon-
strate the suitability of the proposed method to selectively deter-
mine VFAs in wastewater in terms of recovery, sensitivity and ac-
curacy. Likewise, as mentioned earlier, the AD sample was used to
test the accuracy of the method, showing good precision in mea-
surements within the same day and on 5 different days. Therefore,
the applicability of the proposed method to this type of samples is
validated.
The performance of the proposed method is comparable with
other extraction methods for VFAs determination in wastewater

6
M.A. Vargas-Muñoz, V. Cerdà, L.S. Cadavid-Rodríguez et al.
Fig. 3. Chromatogram of the VFAs found in the fermenter reactor sample at pH 7.0 on day 10. (1) Acetic acid, (2) Propionic acid, (3) Isobutyric acid, (4) Butyric acid, (5)
Isovaleric acid, (6) Valeric acid, (7) Isocaproic acid, (8) Caproic acid and (IS) Internal standard: 2 ethylbutyric acid.
(Table S3). Respect to manual LLE methods [11,15,30,39,40], the ex-
tractant solvent consumption is reduced from 5 to 50 times, mak-
ing this process more economical and environmentally friendly.
Likewise, the extraction of the target analytes into few microliters
of extracting solvent favors the preconcentration of those VFAs
present at low concentrations. Other extraction methods achieved
lower LOD; however, this involved the use of sequential extrac-
tions accompanied by mass spectrometry detection [11], or long
HS-SPME methods carried out manually [41,42]. Moreover, SPME
requires careful handling due to the fiber fragility; otherwise its
durability and extraction efficiency are affected. [12]. The method
also is versatile because it can be applied to samples with high
concentration, making a dilution of organic drop in TBME, if is nec-
essary. In-syringe-MSA-DLLME provides precise control of the mea-
surement of the sample volume, TBME volume and organic drop
collected, due to the use of a 16,0 0 0 step motor in the burette. In
the same way, extraction in a closed system in a syringe was able
to minimize the loss of acid by volatilization. Thus, the proposed
method is also competitive in terms of precision and reproducibil-
ity.
On the other hand, the fact of carrying out in just 7 min the en-
tire treatment process (sampling, extraction, phase separation, in-
jection of the extract (in vials) and cleaning) means a great time
saver, considering that batch extraction can take up more than 30
min per sample according to the chosen method. In-syringe-MSA-
DLLME system treats up to 8 samples h−1 . The extraction process
(agitation and separation) takes at least 3 min and therefore, it
is possible to simultaneously analyze up to 5 samples h−1 with
the chromatograph, which could make possible an online analysis.
Likewise, it was not necessary to include the centrifugation steps
and a salt drying (Na2 SO4 anhydride), because a good separation
of the organic phase was achieved and the collection of organic
extract in the vial was precise. In contrast with manual SPME,
LLE and vortex-assisted LLE (VALLE), the method analysis time was
significantly shorter. Accordingly, all of the aforementioned allows
this automated method to be positioned as the first to be so close
7
Journal of Chromatography A 1643 (2021) 462034
to a liquid-liquid extraction in batches, without the high volume
demands and the time it requires.
3.3.1. Determination of VFAs in the laboratory biodigester scale
To determine the presence of VFAs in different concentration
ranges, samples of VFAs production were taken of anaerobic fer-
mentation reactors with fish waste at three pH units (7.0; 9.0 and
11.0). The pH of a biodigester not only influences methane produc-
tion but also has an important inhibition effect on acid producing
microorganisms. Fig. S3 of support information shows the lowest
VFAs production at pH 11.0, otherwise the neutral-slightly alkaline
region (pH 7.0-9.0) favors the production of organic acids in quan-
tity and variety. These results are consistent with other works in
which it seems that strong alkaline conditions inhibit the activity
of acidogenic bacteria, as reflected at a pH above 9.0 [43].
3.4. Comparison of proposed automated method vs distillation
method
The most frequently method used for the routine analysis of
VFAs in wastewater samples from anaerobic treatment processes
is distillation (with subsequent titration) [37,44]; found as method
5560 C in standard APHA protocols [33]. In order to make a com-
parison of the proposed method with the distillation method, sam-
ples from fermenter reactor at pH 7.0 (days 1, 3, 5, 7, 10 and
15) were selected and analyzed. Although the total time of pro-
posed method (extraction + separation and detection by GC-FID) is
longer than distillation time; method 5560C only determines TV-
FAs, expressed as milligrams of acetic acid per liter, and consumes
a large volume of reagents and water for its preparation, generat-
ing a large amount of liquid waste.
The methods were compared quantitatively using different sta-
tistical approaches: the Bland-Altman, Deming and Passing-Bablok
tests (Fig. S4). These procedures are adequate to compare the to-
tal concentrations of VFAs, because they are arbitrary to select
which is the dependent and independent variable and consider
M.A. Vargas-Muñoz, V. Cerdà, L.S. Cadavid-Rodríguez et al. Journal of Chromatography A 1643 (2021) 462034

both methods with an experimental error [7]. Deming regression

RR (%)
and the Passing-Bablok regression indicates that there is no signif-

106.2

107.4

102.5
103.5
112.5
102.6
88.3
95.5

98.1

97.3
98.3

97.3

93.3

97.6
96.7
80.1
icant difference between the two methods (α >0.05). The difference
found by Bland-Altman (194 mg L−1 ) can be attributed to various
sources of error that underestimate or overestimate TVFAs concen-

106.2 ± 2.4
Cf (mg L−1 )

125.8± 4.4
trations. Particularly, total concentrations determined by the distil-
47.8 ± 2.2
80.1 ± 0.1

49.2 ± 4.0

53.7 ± 0.8
97.6 ± 1.0

13.3 ± 0.7
23.5 ± 0.3

118.0±7.5
100.0±0.9

65.0± 0.2
9.7 ± 1.0

9.3 ± 0.6

98.7±1.2
96.3±6.0
8.8 ±1.0

lation method were higher than concentrations found with auto-

Valeric

mated method. Factors in the distillation process such as sample

ND

ND

ND
pretreatment, titrant addition rate and magnetic stirring can affect
the accuracy of VFAs monitoring. On the other hand, ionic interfer-
ents (bicarbonate, VFAs, sulphide, phosphate, ammonium and oth-
RR (%)

100.6

108.4

107.6

103.9
94.1
97.5
82.3

96.7

98.2

93.1

98.8

96.3
94.8
92.4
97.0

99,0
ers) and solid interferents (i.e. cellulose, hemicellulose, and other
carbon and phosphorus salts) can react with the titrant, affecting
the titrant consumption and therefore the results [44]. So, distil-

535.9 ± 3.0
545.5 ± 2.1
108.4 ± 2.8
Cf (mg L−1 )

lation method can be empirical and provide an incomplete and

48.8 ± 5.5

48.5 ± 4.2
82.3 ± 0.1

53.8 ± 0.8
98.8 ± 0.2

583.3±1.9
637.9±3.9
100.3±1.1

101.1±0.3

642.0±2.1
9.7 ± 1.8

9.3 ± 0.7
Isovaleric

99.5±5.1
9.4 ±1.8

somewhat variable recovery, requiring a recovery factor for each

apparatus [12,33]. Likewise, the recoveries of this method can de-
ND

ND

ND

crease if individual VFAs are present in very low concentrations.

Finally, the fact of not considering the concentrations of isocaproic
acid in the TVFAs could also influence the total quantification of
RR (%)

112.9
110.5

103.5

100.1
105.9
108.7

109.9
105.5
102.9
93.5

94.4
95.0

94.9
96.3
96.8
99.6

organic acids. In another study carried out in large-scale anaerobic

digestion plants where the titration method was compared with a
GC method, a difference of 873 mg L−1 of TVFAs was found [44].
Thus, compared with In-syringe-MSA-DLLME-GC-FID, lower aver-
887.2± 14.6
839.1 ± 9.2
848.6 ± 3.3
108.7 ± 7.9

105.5 ± 1.6
Cf (mg L−1 )

54.9 ± 4.5
55.0 ± 5.3
93.5 ± 1.0

10.0 ± 2.1
53.0 ± 2.6

945.4±4.2
926.9±4.6
11.3 ±2.2

Ca: concentration added; Cf: concentration found (average ± SD, n= 3), SD: standard desviation (n= 3), RR: relative recovery, ND: No detected.

age differences were provided.

9.4 ± 3.3
91.1±5.1

83.7±1.7

90.7±1.2
Butyric

4. Conclusions
ND

ND

ND

Concentration added of 1 mM is equivalent to 60.1 mg L−1 of Ac, 74.1 mg L−1 of Pr, 88.1 mg L−1 of Ib y Bu, 102.1 mg L−1 of Iv and Va.

A simple, fast and selective method based on in-syringe-MSA-

DLLME was used as sample preparation procedure followed by GC-
RR (%)

110.8
108.2

103.7

120.3

107.7
103.6

116.3
105.1
109.3

111.4
100.4
110.3
91.2

91.5

94.0

99.3

FID for the extraction and determination of volatile fatty acids (C2-
C5) in anaerobic digestion processes. To our knowledge, this is
the first automated method for individual quantification of VFAs
that allows extracting, preconcentration and cleaning at the same
471.7 ± 2.5
483.3 ± 1.6
103.6 ± 1.8

527.4 ± 2.0
Cf (mg L−1 )

109.3 ±6.9
54.1 ± 2.5

52.5 ± 3.1

53.8 ± 4.6
91.2 ± 0.3

12.0 ± 0.7

572.1±3.4
568.7±6.0
11.1 ±2.6
Isobutyric

9.4 ± 1.3

time, avoiding the need to perform multiple extractions in a batch

91.4±3.9

80.6±5.4

87.5±0.9
Application of the proposed In-syringe-MSA-DLLME-GC-FID method to determine VFAs in wastewater samples.

method and the problems derived from the direct aqueous injec-
ND

ND

ND

tion in column. A multivariate and univariate optimization was ap-

plied to find the best analytical conditions in the system. Results
showed tert-butyl methyl ether as the most suitable solvent for
RR (%)

111.1
103.2

112.7

112.4
113.4

111.2
109.5
111.0

110.1

106.2

107.1
108.1
101.8
110.0

extraction. The characteristics of the designed system and the se-

91.3

96.4

lected extraction solvent avoided the centrifugation stage and the

drying of the extract, having a great time saving. The method was
successfully applied for the analysis of complex samples coming
1697.2 ±3.5
110.1 ±11.7

1686.1± 7.0

1739.6 ±3.4
1794.4± 4.2
113.4 ± 2.6
109.5 ± 1.1
Cf (mg L−1 )

1761.5±5.9
11.3 ± 2.0

56.2 ± 1.3
55.0 ± 2.8

from WWTP and anaerobic processes. Sample matrix interference

10.3 ±1.1

9.6 ± 3.2
Propionic

55.6±5.9

67.6±1.3
82.2±9.0

78.7±0.7

was no observed, so the approach presented may be useful for

the routine analysis of VFAs in the control of anaerobic processes
ND

ND

ND

of municipal or industrial SWTPs and WWTPs, being competi-

tive in terms of sensitivity, precision, frequency and solvent sav-
RR (%)

ing, respect to previously informed manual procedures. Likewise,

113.7
111.8

102.9

113.8

113.8
108.4
103.8

109.6

109.5
112.3

104.3
107.9
85.5

91.7
84.5
98.0

the method can be extended to micro scale studies in the labo-

ratory and other complex aqueous matrices (leachate, wetlands).
Finally, the statistical comparison between the distillation method
8055.1 ± 88.5
8064.3 ± 53.4
8097.4 ± 11.6

and the automated method did not have a significant difference in

8159,4 ±18.7
109.1 ± 11.0

8119.9±27.4
108.4 ± 2.0
Cf (mg L−1 )

112.3± 0.9
55.9 ± 0.7

11.4 ± 0.3
54.7 ± 2.2
10.3 ± 0.3
49.0 ± 2.3

the quantification of TVFAs. Therefore, the proposed methodology

11.4 ±1.6

62.3±7.7

51.3±3.1

68.3±2.7

has good accuracy and is reliable for the analysis of organic acids.
Acetic

ND

ND

ND

Credit Author Statement

M.A. Vargas-Muñoz: Research, writing

Ca (mg L−1 )

Víctor Cerdà: Methodology

L.S. Cadavid-Rodríguez: Reviewing, methodology
1 mM∗

1 mM∗

1 mM∗

1 mM∗
100

100

100

100

Edwin Palacio: Conceptualization, methodology, supervision

10
50

10
50

10
50

10
50
0

Declaration of Competing Interest

Sample

The authors declare that they have no known competing finan-

Table 2

AD

cial interests or personal relationships that could have appeared to

FR
O
I

influence the work reported in this paper.

∗

8
M.A. Vargas-Muñoz, V. Cerdà, L.S. Cadavid-Rodríguez et al.
Acknowledgments
The authors acknowledge financial support from the Comuni-
tat Autonoma de les Illes Balears through the Direcció General de
Política Universitaria i Recerca with funds from the Tourist Stay Tax
Law (PRD2018/45) and also to the Govern de les Illes Balears and
the Oficina de Cooperació al Desenvolupament i Solidaritat of the
University of the Balearic Islands (OCDS-CUD2018/1). M.A.V-M. ac-
knowledges the support from the Spanish Ministry of Science, In-
novation and Universities (MCIU) for the pre-doctoral research fel-
lowship (FPU19/06082). E.P. thanks to Emaya for the provision of
wastewater samples and Dr. Sabrina Clavijo for her contribution.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.chroma.2021.462034.
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