ENVIRONMENTAL ENGINEERING SCIENCE ORIGINAL ARTICLE

Volume 00, Number 00, 2017

ª Mary Ann Liebert, Inc.
DOI: 10.1089/ees.2017.0190

New Approach for Determination of Volatile Fatty Acid

in Anaerobic Digester Sample
Biswabandhu Chatterjee,* Loganath Radhakrishnan, and Debabrata Mazumder

Department of Civil Engineering, Environmental Engineering, Indian Institute

of Engineering Science and Technology (IIEST), Shibpur, Howrah, India.
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

Received: May 5, 2017 Accepted in revised form: August 4, 2017

Abstract
Presence of volatile fatty acids (VFAs) is one of the major indicators of anaerobic digestion of organic matters
present in wastewater, activated sludge, organic fractions of municipal solid wastes, and landfill leachates. The
present article focused on a modified spectrophotometric method for measuring VFAs, based on the classical
Montgomery method, and also compared its performance with other existing techniques of VFA determination
such as distillation method, high-performance liquid chromatography (HPLC), and gas chromatography (GC).
Comparisons have been made with regard to measured concentration of acetic acid standards and accuracy of
the respective method. In addition, comparisons between the proposed method and GC, HPLC, and distillation
methods have been made with regard to VFA measurement (as CH3COOH) from real anaerobic samples. The
distillation method showed poor mean accuracy of 74.96% in comparison to that for the HPLC (using Aminex
column) (100.00%) and for the GC (99.81%). The modified spectrophotometric method showed a superior
accuracy range (95.76–115.35%) over five sets of experimental trials. The precision range (1.66–27.63%) of the
proposed methodology was compared to a past case study of a similar spectrophotometric method (1.70–
14.00%) along with the GC method (5.70–14.80%) and the distillation method (1.80–4.90%). In addition, limit
of quantification and standard deviation were also compared.

Keywords: anaerobic digester sample; comparative study; modified spectrophotometric method; quantification;
volatile fatty acid

Introduction observed that under normal conditions involving the anaer-

obic biodegradation of organic matters, the VFAs are con-
Importance of volatile fatty acid
verted into carbon dioxide and methane, which increases the

A naerobic digestion (AD) of organic matters leads to

the formation of volatile fatty acids (VFAs), which are
an indication of bacterial activity in a sample matrix. These
system pH. This eventually results in the formation of more
and more VFAs due to breakdown of the simple monomers,
thereby countering the increase in pH. Acidogenesis pri-
low molecular mass carboxylic acids (C2-C7 monocarboxylic marily accounts for the formation of the VFAs, and it can
aliphatic acids) or short chain fatty acids (SCFAs) are im- be defined as a process in which simple soluble organic mat-
portant intermediates and metabolites (Siedlecka et al., 2008) ters or monomers are converted to acids and alcohols of
in biological processes involving the AD of wastewater short chains by pH-insensitive acidogenic bacteria. In an-
(Narkis et al., 1980), activated sludge (Albaiges et al., 1986; aerobic digesters, the VFAs are mainly acetic, propionic,
Lie and Welander, 1997), organic fraction of municipal solid butyric, and valeric acids. The subsequent conversion of
waste (OFMSW) (Chatterjee and Mazumder, 2016), and these VFAs and alcohols into methane and carbon dioxide
landfill leachates (Montgomery et al., 1962; Yan and Jen, by syntrophic acetogens and methanogenic bacteria is termed
1992; Manni and Caron, 1995). Salts of lower chain fatty as methanogenesis.
acids or VFAs are formed during the AD of the aforemen- In the measurement of VFA from wastewater, activated
tioned organic matters (Yang and Choong, 2001). It has been sludge, OFMSW, and landfill leachates, the following factors
play a pivotal part: accuracy, precision, limit of quantification
(LOQ), recovery of the weak acids as individual acid or as
*Corresponding author: Department of Civil Engineering, En- acetic acid and rapidity. In addition, the operational ease and
vironmental Engineering, Indian Institute of Engineering Science
and Technology (IIEST), Shibpur, Howrah 711103, West Bengal, technoeconomic viability, in terms of chemicals needed, are
India. Phone: +91 8335916919, +91 9433501315; Fax: +91 33 equally crucial. Analysis of VFA is significant as a quality
2668 2916/4564; E-mail: cbiswabandhu@yahoo.com assurance index in some foods (Tangerman and Nagengast,

1
 2 CHATTERJEE ET AL.

1996). Studies involving the intestinal tract and health also NaOH solution (Siedlecka et al., 2008), was modified later on
analyze for VFAs (Randall et al., 1997). Apart from these, by replacement with the potentiometric titration system. In
determination of VFAs is pertinent in the biological removal case of the potentiometric system of titration since the pH
of nitrogen and phosphorus from wastewater (Eilersen et al., range varied from 4.00 to 7.00, a conversion factor was as-
1994; Raposo et al., 2013) and in the nitrification/denitrifi- signed depending on the assumed concentration of the weak
cation within activated sludge (Wentzel et al., 1991; Elefsi- acid present in the aqueous sample. The conversion factors,
niotis and Wareham, 2007). The understanding of the VFA as such, also varied with different concentrations of weak
profile and the complete mechanism by which the VFAs in acid since their degree of ionization depended on concen-
wastewater/activated sludge function is still unknown at trations; for example, two different conversion factors were
large. However, it can be assumed from different studies that reported to have been used for acetic acid concentrations
the VFAs in wastewater act as a carbon source (Buswell greater and less than 250 mg/L in the region of pH 7.00 (Yang
et al., 1960) for the proper functioning of various nitrifying and Choong, 2001).
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

and denitrifying bacteria, that is, the VFAs provide the re-
quired energy for carrying out the nitrification/denitrification. Titrimetric methods. Titration methods involving the de-
The reduction in pH brought about by the VFAs affects the termination of VFAs from anaerobic digester sample included
storage stability of waste incineration residues and also in- the simple titration step, where total VFA and bicarbonate
creases the mobility of heavy metals and radionuclides. The concentrations from anaerobic digester samples were mea-
generation of unpleasant odor in various organic waste sured over a pH range of 5.5–7.65, proposed by Anderson and
matters, such as wastewater and municipal solid wastes, is Yang (1992), and the five-point titration procedure, proposed
due to one of the chemical classes (mostly butyric acid) by Moosbrugger et al. (1993). VFA determination in anaer-
constituting VFAs (Siedlecka et al., 2008). Thus, it is im- obic digester samples by the titrimetric method is very simple
perative that the VFA measurement techniques should be but the involvement of an empirical factor while calculating
such that they do not involve indirect, time-consuming, and the concentrations of VFA deemed this method to be rigorous
material-demanding analysis but are amenable to field ap- compared to the proposed methodology. The empirical factor
plications. In addition to that, repeatability of results, lower is largely influenced by the effect of the activity coefficients of
LOQ, and an optimum cost aspect are also needed. three different ionic species (H+, HCO3-, and CH3COO-)
present in an anaerobic digester sample. In addition to the
Brief overview on existing methods aforesaid titrimetric methods for monitoring of VFAs from
of VFA determination anaerobic digester samples, Lahav and Morgan (2004) gave a
detailed, critical, and comparative evaluation of other popular
So far, the various techniques for measuring VFAs include titrimetric methods that encompass analysis of VFAs from
distillation method, titrimetric methods (simple titration meth- anaerobic digester samples.
od and five-point titration method), Montgomery method
(Montgomery et al., 1962), gas chromatography (GC), high- Montgomery method. One of the classical VFA mea-
performance liquid chromatography (HPLC), gas-liquid surement techniques is the Montgomery method (Montgomery
chromatography (GLC), paper chromatography (Mueller et al., et al., 1962). This classical method of colorimetric composite
1956), capillary chromatography (Fischer, 2002), headspace determination of the VFA (Montgomery et al., 1962) had rel-
chromatography (Boe et al., 2007), online spectrofluorimetric atively convenient and precise steps for the preparation of the
analysis via a multisyringe/multipumping combined system esters in comparison to the distillation method. However, the
(Palacio-Barco et al., 2010), fluorimetric method (Robert- involvement of colorimeter in measuring the optical density of
Peillard et al., 2009), and spectrophotometric method. In the prepared samples/standards caused the method to be less
most of these techniques, numerous problems were encoun- accurate and more cumbersome compared to the spectropho-
tered, because of which method development for VFA tometric method. Unlike the Montgomery method, in case of
analysis is still an ongoing area of research. The application the modified spectrophotometric method, the absorbance of the
of these methods for determining VFA is largely subject to sample/standard can be directly measured using a single/double
variation in the types of sample, availability of apparatus/ beam spectrophotometer.
chemicals (or reagents)/man power, and extent of accuracy,
as well as sensitivity desired. Paper chromatography method. Another classical meth-
od for VFA determination is the chromatographic method
Distillation method. The distillation method of VFA de- developed by Bulen et al. (1952). Later on, Mueller et al.
termination (Greenberg et al., 1992) was mainly opted for the (1956) developed this method, by modifying the column length
routine determination of VFAs in wastewater and the routine and solvent system, to completely recover the lower volatile
control of sludge digestion. In the distillation method of VFA acids by using a single eluting agent. However, the modified
determination, large volumes of samples are required thus method lacked reproducibility of the results. A similar paper-
making it tedious as well as cumbersome. One of the major chromatographic procedure developed by Buswell et al. (1960)
problems associated with the distillation method is that the produced results that were mostly semiquantitative.
recovery of acetic acid is particularly difficult since it does
not form an azeotropic mixture with water (Yang and HPLC method. Determination of VFAs in anaerobic di-
Choong, 2001). Apart from that, the proportions of individual gester sample via HPLC is a reliable, quick, and efficient
VFAs recovered depend on the experimental conditions. The technique, which produces reproducible and consistent re-
distillation method, in which the contents of the volatile acids sults. Not only that, the preparation of sample is very easy
in the steam distillate is determined by titration with 0.1 N involving centrifugation and filtration of the raw sample
 VFA MEASUREMENT VIA SPECTROPHOTOMETRIC METHOD 3

followed by the analysis of the prepared sample directly. et al., 2009; Palacio-Barco et al., 2010), which allows the se-
However, HPLC involves expensive setup, which is exclu- lective determination of VFA, in a range of 19–1,000 mg/L,
sively meant for measuring the VFA content in an aqueous with a frequency analysis of nine samples per hour, but involves
sample. The determination is largely dependent on the an even more expensive setup and demands trained operational
availability of certain cation exchange columns, which are skills, similar to the GC and the HPLC methods.
specifically designed to measure/separate the VFAs. So far, Compared to all the above processes, the proposed modified
three different cation exchange columns have been reported spectrophotometric method for VFA (as mg/L CH3COOH)
to measure the VFAs via HPLC and these are Supelcogel determination is not only easier but also involves a lower cost in
610H, Aminex HPX87H, and ORH 801. The working of each terms of setup and chemical usage. Besides, the method is fast
column is based on a unique principle. The ORH 801 column and also gives reproducible results, which are accurate and
has the ability to withstand extreme pH conditions (pH: 0–14) have a wide LOQ. This article basically highlights the detailed
and separates VFAs according to their respective pKa values. methodology involving a new approach for VFA determination
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

Thus, this column has been reported to be used for the de- in anaerobic digester samples. This new method of VFA de-
termination of VFAs from fecal samples (Chen and Lifschitz, termination is a modified spectrometric procedure based on the
1989). The Supelcogel 610 H column has been used for an- classical Montgomery method (Montgomery et al., 1962). The
alyzing VFAs from pig slurry, where the six different SCFAs modifications lie in the preparation of a reagent that played a
(acetic, propionic, isobutyric, butyric, isovaleric, and valeric crucial role in controlling the final pH and color development.
acids) were reported to be isolated with this column using an This was accomplished since the Montgomery method did not
isocratic phosphate eluant coupled with an ultraviolet de- provide any control over the pH and hence the color develop-
tector in series (Peu et al., 2004). ment was not consistent. As such, the proposed method satis-
The first reported determination of VFA via HPLC was fied the conditions mentioned in the Montgomery method in
carried out by Guerrant et al. (1982). The team analyzed a terms of the desired working pH range. The article also briefly
standard mixture of 25 SCFAs produced by anaerobic bacteria highlights the approach of some of the existing popular meth-
in culture media by HPLC equipped with Aminex HPX-87 ods for VFA measurement, the comparison between methods
column (cation exchange column). Later, Chen and Lifschitz of distillation, GC, and HPLC with the proposed method, and
(1989) determined VFAs derived from fecal samples via also justifies the need for selecting the proposed method over
GLC, and HPLC equipped with ORH-801 organic acid the popular methods.
chromatographic column (cation exchange column). It was Comparisons between the proposed methodology and the
reported that similar to the Aminex HPX87H column, the distillation method, the GC method, and the ultra-HPLC
ORH-801 (sulfonated polystyrene divinylbenzene in the hy- (equipped with C18 column) method were attempted using
drogen form) column had the capacity to separate only the anaerobic digester samples of unknown acetic acid concentra-
VFAs. Efficiency in terms of quick sample preparation, direct tions, obtained from a laboratory-scale three-stage mesophilic
analysis, and quick separation of the VFAs along with traces AD system comprising a hydrolytic chamber, an acidogenic
of the nonvolatile dicarboxylic and keto acids was noticed in chamber, and a methanogenic chamber, treating fruit and veg-
both the experimental studies, while measuring via HPLC. etable waste, a laboratory-scale hybrid up-flow anaerobic sludge
The accuracy and precision of VFA measurement via HPLC blanket (HUASB) digester treating slaughter-house wastewater,
was vindicated in a study by Peu et al. (2004), who proposed a and a pilot-scale floating dome-type digester treating OFMSW.
method to determine VFA content in pig slurry sample using
HPLC. However, the involvement of expensive column, ex- Experimental
clusively meant for determining VFA, mostly made the pro-
Materials and reagents
cess economically infeasible.
For the purpose of preparing the synthetic samples/standards,
GC method. In the GC method of VFA determination, which were used for calibrating the GC, the HPLC (equipped
measurement of individual VFA concentration was possible by with liquid chromatography [LC]-AMINO column), and the
means of a set of calibration curves obtained using five aqueous modified spectrophotometer, glacial acetic acid (assay 99.7%) of
solutions of acetic acid, propionic acid, butyric acid, valeric strength 60.1 g/L and triple distilled water were used. In case of
acid, and caproic acid in the concentration range of 5–1,000 mg/ the modified spectrophotometric method, sample preparation
L. The preparations of the standards as well as the samples were necessitated the usage of the following chemicals of analytical
based on the Manni and Caron’s (1995) procedure. However, grade, without further purification: glacial acetic acid (EM-
the present study focused on the measurement of acetic acid PARTA; Merck made), ethylene glycol (EMPARTA; Merck
concentrations from anaerobic digester samples. For this pur- made), sulfuric acid (about 98% pure EMPARTA; Merck
pose, calibration curves were developed using 14 different made), hydroxylamine hydrochloride (99% pure; Loba Chemie),
standards of acetic acid concentrations ranging from 100 to sodium hydroxide pellets (EMPLURA, Merck made), and ferric
1,400 mg/L. The GC method of VFA determination has proved chloride [anhydrous Iron (III) Chloride EMPLURA, Merck
to be comparatively more precise and accurate, compared to the made]. Tripled distilled water was used entirely in all the anal-
aforementioned methods, with provision for measuring indi- ysis. While calibrating the HPLC system, equipped with C18
vidual concentration of VFAs. However, still this technique is column (250 · 4.6 mm i.d.), HPLC-grade acetic acid (assay
seemingly not applicable to sludge liquor and similar to HPLC ‡99.8%), manufactured by Sigma-Aldrich, and HPLC-grade
also involves a costly setup. water were used for preparing the following standards: 62.5, 125,
250, 500, 750, 1,000, and 2,000 mg/L.
Other methods. Another VFA measurement technique Raw samples from the five anaerobic chambers were
includes the online spectrofluorimetric system (Robert-Peillard centrifuged at 3,000 rpm for 5 min, following collection.
 4 CHATTERJEE ET AL.

FIG. 1. Measured VFA concen-

trations (in mg/L as CH3COOH)
versus theoretical VFA concentra-
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

tions (in mg/L as CH3COOH) for

distillation method. VFA, volatile
fatty acid.

Thereafter, the samples were filtered using 11-cm-diameter (grade No. 42). The analysis via distillation method was
commercial-grade filter paper of pore size 50 lm. Following performed using only the filtered anaerobic samples.
filtration, the filtered samples were diluted with commer-
cially available distilled water for the purpose of testing via
Determination of VFA by distillation method
GC, ultra-HPLC (equipped with C18 column), and modified
spectrophotometric method. For analysis via ultra-HPLC Glacial acetic acid of strength 60.1 g/L and triple distilled
(equipped with C18 column), the abovementioned filtered water were used for preparation of standards ranging from
samples were also tested following dilution with HPLC- concentrations of 100–1,400 mg/L. The standards were then
grade water. In addition, while testing the raw samples in steam distilled as per the procedure of ‘‘Distillation Method’’
ultrahigh-performance liquid chromatography (UHPLC; described in the Standard Methods (American Public Health
equipped with C18 column), further filtration of aforesaid Association and Water Environment Federation, 1995).
filtered samples was carried out using Whatman filter paper Thereafter, the volatile acid content in the distillate was

FIG. 2. Percentage recovery of

VFA concentrations (in mg/L as
CH3COOH) versus theoretical
VFA concentrations (in mg/L
as CH3COOH) for distillation
method.
 Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

5
FIG. 3. HPLC chromatograms for CH3COOH concentrations of (A) 100 mg/L, (B) 400 mg/L, (C) 600 mg/L, (D) 800 mg/L, (E) 1,000 mg/L, and (F) 1,200 mg/L. HPLC, high-
performance liquid chromatography.
 6 CHATTERJEE ET AL.

determined with 0.1 N sodium hydroxide solution and was following dilution with HPLC-grade water; and finally, by
expressed as acetic acid content. measuring the centrifuged and filtered real anaerobic samples
following dilution with commercially available distilled water.
Determination of VFA by high-performance
liquid chromatography Determination of VFA by gas chromatography
Similar to the distillation method, the standards for the Unlike the procedure described by Siedlecka et al. (2008),
HPLC method were acetic acid solutions in triple distilled the standards of VFAs (acetic acid in triple distilled water)
water with concentrations ranging from 100 to 1,400 mg/L. ranging from concentrations of 100–1,400 mg/L were pre-
These were the same standards, which were used for testing via pared using 99.7% glacial acetic acid of strength 60.1 g/L.
the other three methods (GC, modified spectrophotometric The analysis of the fourteen standards and the five anaerobic
method, and distillation method). The analysis of these stan- digester samples was carried out using the Agilent 7890B GC
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

dards was performed using the Shimadzu LC-2010 HPLC system and the YL 6100 GC system. Both the GC systems
system packed with a cation-exchange HPLC column (SU- were equipped with ‘‘SUPELCOWAX 10’’ fused silica
PELCOSIL LC-AMINO, length of 30 cm and internal diameter capillary GC column (30 m · 0.25 mm, 0.25 lm). ‘‘Flame
of 5 mm). The wavelength setting was 210 nm, the injection ionization detector (FID)’’ was used for the detection of the
volume was 20 lL, and the run time was 10 min. Mobile phase VFA. The back inlet temperature was set at 260C with a split
selected was similar to that mentioned in Guerrant et al. (1982). ratio of 30:1. The injection volume was set to 1 lL. The front
VFA concentrations were determined by comparing the peak detector temperature was set at 270C. The oven temperature
area of calibration with that of the sample. was initially set at 80C, with a hold time of 0 min and ramp
However, the analysis of the five anaerobic digester sam- of 10C, and the final temperature was 250C. EZChrom Elite
ples containing unknown acetic acid concentrations was done Compact system was used as the data analysis system.
using an UHPLC system, where the chromatographic separa- Standard/synthetic samples were directly subjected to
tion was performed using isocratic elution. The run time was set GC-FID, whereas 5 mL of the real anaerobic sample was
at 6 min. The column oven temperature, injection volume, and extracted with 5 mL of dichloromethane, and the resultant
wavelength were according to that mentioned in Mitra et al. solvent was subjected to GC-FID. The sample extraction
(2016). The mobile phase was a buffer of 20 mM NaH2PO4 in procedures were similar for both the aforesaid GC systems.
HPLC-grade water with pH 2.2. Hitherto, C18 column was Similar to HPLC, concentration was determined by com-
never reported to analyze VFA concentrations (as CH3COOH) paring the peak area of calibration and the peak area of the
in anaerobic digester samples. Hence, to ascertain the consis- sample. The quality of the column used in a GC system is
tency of the testing via the UHPLC equipped with C18 column, extremely crucial for the precise analysis of the VFAs
the centrifuged and filtered real anaerobic samples were ana- present in an aqueous sample. This is because the polarity
lyzed in three different ways: first, by measuring the centri- of the column stationary phase is mostly critical for the
fuged and filtered real anaerobic samples directly following successful separation of the VFAs. Improved peak resolu-
filtration using Whatman 42-grade filter paper; second, by tions can only be obtained if the polarity of the column
measuring the centrifuged and filtered real anaerobic samples stationary phase matches closely with the polarity of the

FIG. 4. Peak area count versus

theoretical VFA concentrations (in
mg/L as CH3COOH) for HPLC
method.
 FIG. 5. Measured VFA concen-
trations (in mg/L as CH3COOH)
versus theoretical VFA concentra-
tions (in mg/L as CH3COOH) for
HPLC method.
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

FIG. 6. Chromatograms for CH3COOH concentrations of (A) 0.125 mg/mL, (B) acidogenic sample, (C) hydrolytic
sample, (D) methanogenic sample, (E) floating dome-type digester sample, and (F) HUASB digester sample (all the digester
samples were 10 times diluted with commercial-grade distilled water). HUASB, hybrid up-flow anaerobic sludge blanket.

7
 8 CHATTERJEE ET AL.

VFAs (Zhang et al., 2015). Prolonged use of a particular of ester was found to be highest after 2 min and found to
GC column, for analysis of SCFAs, without repair may lead decay slowly after 4 min. Thereafter, 0.5 mL of 10% hy-
to the degradation of the column through acid-base reac- droxylamine hydrochloride (HONH3Cl) was added, followed
tions with the stationary phase. by the addition of 2 mL of 4.5 N NaOH solution and 10 mL of
10% FeCl3 solution (acidified with H2SO4). It was ensured
Determination of VFA by modified that the pH of the resultant mixture remained between 1.2 and
spectrophotometric method 2 since it was observed that the stability of the brown color
was enhanced between the aforesaid pH ranges. Finally, the
Approach of the modified spectrophotometric method. The
content in the test tube was diluted to 49.7 mL before the
proposed methodology is based on the well-known colori-
measurement of absorbance at 495 nm using single/double
metric ferric hydroxamate method for determination of car-
beam spectrophotometer.
boxylic esters. Esterification is carried out by ethylene glycol,
because it gives good yields of ester and low blank values.
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

The simplest objective is to use a large excess of nonvolatile Results and Discussion
alcohol that is miscible using water in all proportions with
sulfuric acid as catalyst. Distillation method
Standards of VFA concentrations, ranging from 100 to
Procedure for determination of VFA. For standard/sam- 1,400 mg/L, were initially prepared by dissolving glacial
ple preparation, 0.5 mL of aqueous synthetic sample/anaer- acetic acid in water, as mentioned earlier. Thereafter, those
obic diluted sample was taken into a dry test tube. This was were steam distilled as per the procedure of ‘‘Distillation
followed by the addition of 1.5 mL ethylene glycol and Method’’ described in the Standard Methods (American
0.2 mL of 19.2 N H2SO4. Thereafter, the mixture was heated Public Health Association and Water Environment Fed-
for 3 min in a boiling bath followed by immediate cooling of eration, 1995). The recovered concentrations of the indi-
the mixture. The 3-min duration was chosen, since the yield vidual VFAs were noted and were represented in mg/L

FIG. 7. Chromatograms for CH3COOH concentrations of (A) 0.750 mg/mL, (B) acidogenic sample, (C) hydrolytic
sample, (D) methanogenic sample, (E) floating dome-type digester sample, and (F) HUASB digester sample (all the digester
samples were 10 times diluted with HPLC-grade distilled water).
 VFA MEASUREMENT VIA SPECTROPHOTOMETRIC METHOD 9

as CH3COOH. The recovery factor for each standard was there was a large requirement of the sample volume for VFA
also calculated from the ratio of the measured and the determination, making it cumbersome and tedious. Also,
theoretical concentrations, accordingly. Subsequently, the the proportions of individual volatile acids recovered de-
measured concentrations of CH3COOH solution were pended on the experimental conditions, for instance, acetic
plotted with respect to the theoretical concentrations of the acid is particularly difficult to recover through this tech-
same to find out the mean recovery factor as shown in nique (distillation) as it does not form an azeotropic mixture
Fig. 1. with water.
Values of percentage recovery were also plotted with re- With poor recovery of the acetic acid arising out of the
spect to the theoretical concentrations of CH3COOH as conventional titration method, following isolation of the
shown in Fig. 2. SCFAs by steam distillation, the appropriate substitution was
It was reported in the study by Siedlecka et al. (2008) that brought about by the introduction of the potentiometric ti-
in case of the distillation method the precision ranged be- tration system. The potentiometric titration method showed
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

tween 1.8% and 4.9%. However, on repeating the distillation considerable improvement in terms of absolute recovery of
process for determining VFA, it was observed by the authors the acetic acid, in the range 11–1,164 mg/L (LOQ) with ac-
that the absolute recovery of acetic acid improved from the curacy being above 97% and precision being in the range 1.8–
range of 110 mg/L (LOQ) to 636 mg/L and from the range of 15% (Siedlecka et al., 2008). However, the major drawback
100 mg/L (LOQ) to 1,400 mg/L, with the accuracy showing of this method (distillation followed by potentiometric titra-
improvement from the range of 53–58.2% to the range of 68– tion) was that it required a conversion factor for various pH
85%. Figure 2 reveals that the mean recovery factor in case of ranges, in between 4.00 and 7.00, when the titration was done.
distillation method is about 74.96%, for CH3COOH con- Assigning this conversion factor corresponding to different
centration in the range of 100–1,400 mg/L. Despite the im- pH ranges depended on the assumed concentration of the
provement from the earlier study, as highlighted by the weak acid present in the aqueous sample. The assumed con-
authors, the comparatively poor recovery/accuracy over the centrations of the weak acid varied with the actual concen-
spectrophotometric method deemed the distillation method trations of weak acid, since their degree of ionization was
to be inferior. Moreover, in case of the distillation method, dependent on the concentrations.

FIG. 8. Chromatograms for CH3COOH concentrations of (A) 2 mg/mL, (B) hydrolytic sample, (C) methanogenic sample,
(D) acidogenic sample, (E) floating dome-type digester sample, and (F) HUASB digester sample (centrifuged digester
samples were used after filtering through commercial-grade filter paper and Whatman 42-grade filter paper).
 Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

10
FIG. 9. GC chromatograms for CH3COOH concentrations of (A) 100 mg/L, (B) 500 mg/L, (C) 600 mg/L, (D) 1,000 mg/L, and (E) 1,200 mg/L (Agilent 7890B GC system).
GC, gas chromatography.
 VFA MEASUREMENT VIA SPECTROPHOTOMETRIC METHOD 11

FIG. 10. Peak area count versus

theoretical VFA concentrations (in
mg/L as CH3COOH) for GC
method.
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

High-performance liquid chromatography been plotted with respect to theoretical concentrations of the
same, as shown in Fig. 5.
HPLC chromatograms developed for six typical concen- The mean accuracy level of HPLC method is thus observed
trations of acetic acid using the Shimadzu LC-2010 HPLC as 100%.
system packed with SUPELCOSIL LC-AMINO are shown in For the purpose of measuring the acetic acid concentra-
Fig. 3. tions in the five digester samples, UHPLC equipped with
The peak area for each concentration was noted, and a C18 column was used. Seven standards of acetic acid con-
calibration curve between the theoretical concentration and centrations 62.5, 125, 250, 500, 750, 1,000, and 2,000 mg/L
the peak area, obtained for individual concentration, was also were used for calibrating the system and for developing
prepared, as shown in Fig. 4. the calibration curve. Figures 6–8 show the chromatograms
To find out the accuracy of HPLC method (using the Shi- for standards 125, 750, and 2,000 mg/L along with the
madzu LC-2010 HPLC system packed with SUPELCOSIL chromatograms of the 5 anaerobic digester samples, which
LC-AMINO), measured concentrations of CH3COOH have were diluted 10 times with distilled water and HPLC-grade

FIG. 11. Measured VFA con-

centrations (in mg/L as
CH3COOH) versus theoretical
VFA concentrations (in mg/L as
CH3COOH) for GC system.
 Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

12
FIG. 12. GC chromatograms of the (A) hydrolytic digester sample, (B) acidogenic digester sample, (C) methanogenic digester sample, and (D) floating dome-type digester
sample (using Agilent 7890B GC system).
 Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

13
FIG. 13. GC chromatograms of the (A) 100 mg/L, (B) 500 mg/L, (C) 600 mg/L, (D) 1,000 mg/L, (E) 1,200 mg/L, and (F) HUASB digester sample (using YL 6100 GC system).
 14 CHATTERJEE ET AL.

water for the first 2 runs and without any dilution for the tion, where VFA analysis is not a routine practice, these
last run. columns are likely unavailable. Alternatively, single/dou-
The UHPLC system equipped with the C18 column was ble beam spectrophotometers are ubiquitous and sufficient
calibrated using seven standards of acetic acid concentrations for VFA analysis.
(prepared with HPLC-grade acetic acid and HPLC-grade
water) 62.5, 125, 250, 500, 750, 1,000, and 2,000 mg/L and
Gas chromatography
the corresponding calibration curve, with R2 value 0.999, was
developed. The equation obtained was y = 0.0085x, where y is Standard/synthetic samples of acetic acid concentrations
the peak area count and x is the acetic acid concentration ranging from 100 to 1,400 mg/L were tested in two separate
in mg/L. GC systems for the purpose of calibrating the systems and
It can be clearly observed from Figs. 6–8 that UHPLC for developing the calibration curves. In the Agilent 7890B
systems equipped with C18 columns are not best suited for GC system, diluted samples from the hydrolytic, the
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

measuring acetic acid or any other SCFAs present in an- acidogenic, the methanogenic, and the floating dome-type
aerobic digester samples. The standard/synthetic samples digester were tested. The sample from the HUASB digester
(concentrations between 100 and 1,400 mg/L) prepared with was tested in the YL 6100 GC system. The GC chromato-
glacial acetic acid (assay 99.7%; EMPARTA; Merck made) grams of five typical standard CH3COOH concentrations
and triple-distilled water, which were detectable via the GC, obtained using the Agilent 7890B GC system are shown in
and the HPLC equipped with AMINO column, could not be Fig. 9.
detected using the UHPLC system equipped with the C18 The corresponding calibration curve prepared between the
column. Even though the particle sizes (5 lm) in both the theoretical concentrations of CH3COOH and the peak area
columns used were similar, the variation in results may have count, obtained for the above five standard concentrations,
been due to the change in the HPLC systems. The obvious for Agilent 7890B GC system, is shown in Fig. 10.
variations in the LC techniques of the two instruments may To find out the accuracy of the GC method (for Agilent
be responsible for the observed differences in results, and 7890B GC system), the measured CH3COOH concentrations
subject to further detailed investigation. In addition, there have been plotted with respect to the theoretical concentra-
is a dearth of literature on measurement of SCFAs from tions of CH3COOH standards as appended in Fig. 11.
anaerobic digester samples using either UHPLC or C18 Mean accuracy of VFA (as CH3COOH) determination by
columns. However, UHPLC systems equipped with C18 GC method is observed as 99.81% from Fig. 11.
column have been reported to measure VFAs from food and The analysis of the diluted samples (10 times diluted with
plant products (Mitra et al., 2016). triple-distilled water) obtained from the hydrolytic, the acido-
Even though measurement of VFAs via HPLC is quick, genic, the methanogenic and the floating-dome type digesters
consistent, and efficient compared to the spectrophoto- was carried out using the same YL 6100 GC system, where the
metric method, it can be said that unless any of the previ- 14 different standards of acetic acid concentrations were tested.
ously mentioned columns (Supelcogel 610H, Supelcosil The corresponding chromatograms of the four digester samples
LC-AMINO, Aminex HPX87H and ORH 801) are avail- are shown in Fig. 12.
able the measurement cannot be done. These columns are The sample from the HUASB digester was analyzed in
not only expensive but also demand specific operative skills the YL 6100 GC system. Similar to the four other digester
to run. In laboratories with available HPLC instrumenta- samples, the HUASB digester sample was diluted 10 times

FIG. 14. Absorbance versus the-

oretical VFA concentrations (in
mg/L as CH3COOH) for the mod-
ified spectrophotometric method.
 VFA MEASUREMENT VIA SPECTROPHOTOMETRIC METHOD 15

1,398.12

40.92
2.93

99.87
with triple-distilled water. The YL 6100 GC system was

1,400
calibrated using five standards of acetic acid concentrations
100, 500, 600, 1,000, and 1,200 mg/L and the corresponding
calibration curve, with R2 value 0.996, was developed. The
equation obtained was y = 0.6322x, where y is the peak area

1,282.01

54.78
4.27

98.62
count and x is the acetic acid concentration in mg/L. Fig-

1,300
ure 13 shows the chromatograms for the five standards of
acetic acid concentrations, and the HUASB digester sample
corresponding to the YL 6100 GC system.

1,149.07

97.58
8.49

95.76
It has been observed that determination of VFAs via GC is the

1,200
most reliable among the aforementioned methods with regard to
LOQ, precision, accuracy, sample preparation, and volume of the
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

Statistical Analysis of Volatile Fatty Acid concentration (mg/L as CH3COOH)

sample required for measurement. Also, the method allows for

1,071.65

55.41
5.17

97.42
measurement of individual VFA concentration. In the study by

1,100
Siedlecka et al. (2008), the precision range for measuring VFAs
via GC was reported to be between 5.7% and 14.8%, and the
absolute recovery of acetic acid was in the range of 4.6 mg/L
(LOQ) to 1,000.7 mg/L with accuracy between 92% and 100.7%.

991.93

13.69

99.19
1.38
It was also reported that only 2 mL of the aqueous solution was

1,000
needed for the testing. However, the GC method necessitated the
preparation of diazomethane, a derivative’s agent, which is
considered to be toxic and nonstable. In the present study too,

913.07

15.11
1.66

101.45
extraction of the real anaerobic samples had to be done with

900
dichloromethane, which is highly volatile in nature, before test-

VFA concentration (mg/L as CH3COOH)

ing. Another drawback was the selectivity problem with the
determination of acetic acid, in case of additional volatile con-

50.57
6.07

104.18
833.4
taminants present in the sample that was analyzed. Therefore,

800
even though the GC method can be regarded as the most reliable
of all the methods, including the existing spectrophotometric

748.88

47.67
6.37

106.98
method, for measurement of low VFA concentrations and
700
practical applicability onto surface-water and wastewater sam-
ples, it is yet to give satisfactory results for samples of digester
sludge liquor. This problem was eliminated by the spectropho-
656.07

40.93

109.35
6.24
tometric method.
600

RSD, relative standard deviation; SD, standard duration; VFA, volatile fatty acid.
Modified spectrophotometric method
516.69

103.34
58.72
11.37

Data obtained from modified spectrophotometric method (No. of sets: 5).

500

The earlier spectrophotometric method for VFA deter-

mination produced repeatable results, good accuracy, pre-
391.75

27.18
6.94

97.94

cision, and lower LOQ. The precision and accuracy of the

spectrophotometric method were in the range of 1.3–14%
400

and 82.1–104.2%, respectively, for acetic acid concentra-

tions between 28 and 450 mg/L with absolute recovery
303.03

32.82
10.83

101.01

of acetic acid being in the range of 23 and 457 mg/L. The

300

proposed spectrophotometric method eliminated incon-

sistencies and showed reproducible results. Similar to the
Table 1.

spectrophotometric method, the proposed method has several

223.34

61.71
27.63

111.67

advantages over the other popular methods, such as small

200

volume of sample requirement (0.5 mL) and direct determi-

nation of the VFA (in mg/L as CH3COOH) from the sample.
115.35

24.68
21.39

115.35

Also, rapid multiple determination is possible since analysis

of a single sample takes <15 min.
100

This proposed method substantiates the underlying

principle of esterification of carboxylic acids present in
VFA concentration

digester liquor and the subsequent determination of those

RSD (precision)
(AVG, mg/L)

esters by the ferric hydroxamate reaction, which is in line

Accuracy (%)

with the classical colorimetric method. To measure the

optical density (in this case absorbance), a double/single
(mg/L)
Average

beam spectrophotometer was used instead of a colorimeter

(%)

thereby increasing the accuracy of the measurement. The

SD

absorbance values as measured for the standard samples

 16 CHATTERJEE ET AL.

Table 2. Comparison Between Modified Spectrophotometric Method (Proposed) and Other Existing
Techniques for Volatile Fatty Acid Determination
LOQ as mg/L
Method Accuracy (%) of CH3COOH Precision (%)
Distillation method (Siedlecka et al., 2008) 53–58.2 110 —
Distillation method 68.5–85.0 100 —
Potentiometric titration method (Siedlecka et al., 2008) >97 11 1.8–15
Gas chromatographic method (Siedlecka et al., 2008) >92 5 5.7–14.8
Gas chromatography 89–106 — —
HPLC 93.91–153.18 — —
Spectrophotometric method (Siedlecka et al., 2008) 82.1–104.2 28 1.3–14
Modified spectrophotometric method 95.76–115.35 10 1.38–27.63
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

HPLC, high-performance liquid chromatography; LOQ, limit of quantification.

were plotted with respect to acetic acid concentration as deviation. The accuracy in these two cases is 109% and
shown in Fig. 14. 107%, respectively, whereas the precision is 6.24% and
The concentration data of acetic acid standards are used for 6.37%, respectively. Table 1 demonstrates that the precision
statistical analysis to find out the average, standard duration and the accuracy of the proposed methodology are in the
and relative standard deviation (RSD), as shown in Table 1. range 1.38–27.63% and 95.76–115.35%, respectively, for
The accuracy and precision are estimated for five sets of concentrations of VFAs (mg/L as CH3COOH) between 100
standards having concentrations from 100 to 1,400 mg/L as and 1,400 mg/L.
CH3COOH are also furnished in Table 1. The comparison between the proposed methodology and
For compiling the data in Table 1, the same technique, as the existing ones for VFA determination in terms of accuracy,
mentioned by Siedlecka et al. (2008), has been adopted. precision, and LOQ is presented in Table 2.
RSD has been evaluated for the purpose of determining the While studying the absolute recovery of acetic acid, a
precision of the proposed methodology, whereas the accu- satisfactory range between 100 and 1,400 mg/L was found,
racy has been estimated in percentage by comparing the which can be considered good when compared with other
measured and theoretical concentrations of acetic acid in methods. In addition, Fig. 15 asserts the superiority of the
the samples. It can be observed from the results that all proposed methodology over the other measurement tech-
the average theoretical concentrations of VFA (mg/L as niques as it can be seen that the deviation of results of the
CH3COOH) are within the standard deviation except two measured concentration with respect to the theoretical con-
cases, namely 600 and 700 mg/L. In these two cases, the centration of VFAs is the least in case of the GC method
average values of VFA are found to be out of the standard followed by the proposed method.

FIG. 15. Plotting of measured

and theoretical VFA concentration
(in mg/L as CH3COOH) for modi-
fied spectrophotometric, distilla-
tion, GC, and HPLC methods.
 VFA MEASUREMENT VIA SPECTROPHOTOMETRIC METHOD 17

Table 3. Absorbance Values and Volatile Fatty Acid Concentrations

of Real Anaerobic Samples via Modified Spectrophotometric Method
Floating dome-type
Hydrolytic Acidogenic Methanogenic HUASB anaerobic digester
sample sample sample sample sample
Trial
No. A B C A B C A B C A B C A B C
1 0.524 0.270 1,662 0.353 0.272 926 0.356 0.270 962 0.334 0.270 394 0.361 0.270 548
2 0.452 0.373 0.386 0.336 0.328
3 0.476 0.373 0.394 0.317 0.381
4 0.464 0.381 0.406 0.306 0.295
5 0.501 0.462 0.401 — —
6 0.499 0.412 0.427 — —
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

A, observed absorbance values; B, blank absorbance value; C, calculated concentration (in mg/L as CH3COOH).
HUASB, hybrid up-flow anaerobic sludge blanket.

Moreover, it is possible to analyze as many as 10 samples value for the blank was then subtracted from the average
within a maximum duration of 60 min using the proposed absorbance readings obtained for each of the five anaerobic
method. The accuracy coupled with such optimum analysis samples. Thereafter, the resultant absorbance value of any
time is extremely useful in case of measuring anaerobic particular sample was put into the equation y = 0.0013x, ob-
digester samples, where rapid analysis is very important. tained from Fig. 14, to get the acetic acid concentration (in
This is because, the VFA present in the collected sample mg/L) of that sample. Table 3 gives a detailed report of the
decays very rapidly when exposed to an aerobic environ- VFA concentrations obtained for the five anaerobic samples
ment. LOQ is separately calculated and is found to be via the proposed methodology.
10 mg/L. It can be observed from Table 2 that the proposed Comparisons between GC, UHPLC (using C18 column),
methodology has a superior accuracy range compared to the distillation method, and the proposed method with regard to
distillation method, the HPLC method, and the previous real anaerobic samples were collected from four laboratory-
spectrophotometric method. In addition, the precision range scale anaerobic digesters and one pilot-scale digester, ana-
was also found to be much better in case of the proposed lyses are shown in Table 4.
methodology. Another significant advantage of the pro- The modified spectrophotometric method’s consistent
posed methodology is that it provides a scope for a wider performance with regard to real anaerobic sample analysis
range of acetic acid concentration measurement, between can be clearly observed from Table 4. It can be said that the
10 and 1,400 mg/L, at the aforementioned precision and consistency of the proposed methodology, with regard to real
accuracy. anaerobic sample analysis, is only asserted by the results
Six different absorbance readings (out of nine trials) were obtained by testing via GC and distillation methods. In ad-
considered each for the hydrolytic, acidogenic, and the me- dition, consistency of the proposed methodology, with regard
thanogenic samples and the average was calculated. For the to analysis of standard/synthetic samples prepared using
HUASB and the floating dome-type digester samples, four glacial acetic acid and triple-distilled water, has been also
absorbance readings (out of six trials) were considered and asserted by comparison with GC, HPLC, and distillation
the average absorbance value was calculated. The absorbance methods.

Table 4. Comparison Between Modified Spectrophotometric Method and Gas Chromatography,

Ultrahigh-Performance Liquid Chromatography and Distillation Method
for Volatile Fatty Acid Analysis in Real Anaerobic Samples
Method applied
Modified
spectrophotometric Gas UHPLC
method chromatography (using C18 column) Distillation method
Sample source Measured VFA concentration (in mg/L as CH3COOH)
Hydrolytic digester 1,662 1,647 3,514 1,122 (67.5% recoverya)
Acidogenic digester 926 897 771 660 (71.3% recoverya)
Methanogenic digester 962 970 527 668 (69.4% recoverya)
HUASB digester 394 339 Nil 300 (76.1% recoverya)
Floating dome-type digester 548 541 Nil 363 (66.2% recoverya)
a
Recovery percentages for the distillation method are with respect to results obtained via the modified spectrophotometric method.
UHPLC, ultra high-performance liquid chromatography.
 18 CHATTERJEE ET AL.

Conclusion Chatterjee, B., and Mazumder, D. (2016). Anaerobic digestion

for the stabilization of the organic fraction of municipal solid
Presence of VFAs largely influences the contaminant levels
waste: A review. Environ. Rev. 24, 426.
of various nutrients (inorganic nitrogen and phosphorus) Chen, H.M., and Lifschitz, C.H. (1989). Preparation of fecal
available in wastewater, which in turn disrupts the aquatic samples for assay of volatile fatty acids by gas-liquid chro-
environment to a great deal by causing oxygen depletion, matography and high-performance liquid chromatography.
algal bloom, and eutrophication. The VFAs act as energy Clin. Chem. 35, 74.
suppliers for different bacterial species thereby supporting Eilersen, A.M., Henze, M., and Kloft, I. (1994). Effect of vol-
their growth, which eventually results in an adverse impact on atile fatty acids and trimethylamine on denitrification in
the waterbody. Monitoring of VFAs is thus extremely nec- activated-sludge Water Res. 28, 1329.
essary with regard to maintaining hygienic conditions. A rapid Elefsiniotis, P., and Wareham, D.G. (2007). Utilization patterns
and also dependable method for determining the VFAs is of volatile fatty acids in the denitrification reaction. Enzyme
therefore needed in water quality management. The proposed Microb. Technol. 41, 92.
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.

methodology, that is, the modified spectrophotometric meth- Fischer, K. (2002). Environmental analysis of aliphatic car-
od, offers many advantages over the existing methods of VFA boxylic acids by ion-exclusion chromatography. Anal. Chim.
determination, not only because of its excellent precision, Acta 465, 157.
accuracy, and range of acetic acid recovery but also in terms Guerrant, G.O., Lambert, M.A., and Moss, C.W. (1982). Analysis of
of operation, rapidity, ability to measure VFAs present in short-chain acids from anaerobic bacteria by high-performance
anaerobic digester sludge liquor, leachates, wastewater, and liquid chromatography. J. Clin. Microbiol. 16, 355.
OFMSW. This method is also found to be technoeconomi- Lahav, O., and Morgan, B.E. (2004). Titration methodologies for
cally viable (due to use of low-cost apparatus usage and re- monitoring of anaerobic digestion in developing countries—A
quirement of easily available chemicals) and needs for a small review. J. Chem. Technol. Biotechnol. 79, 1331.
Lie, E., and Welander, T. (1997). A method for determination
volume of the aqueous sample. Moreover, the importance of
of the readily fermentable organic fraction in municipal
the proposed methodology is substantiated by repeatability of
wastewater. Water Res. 31, 1269.
results and consistency with the sample preparation, as illustrated Manni, G., and Caron, F. (1995). Calibration and determination
by the classical Montgomery method. Hence, this method can be of volatile fatty acids in waste leachates by gas chromatog-
inferred to be opted for VFA measurements in places requiring raphy. J. Chromatogr. A 690, 237.
accurate and precise results at low cost and with a limited setup. Mitra, D., Jena, A.K., De, A., Das, M., Das, B., and Samanta, A.
(2016). Prebiotic potential of gum odina and its impact on gut
Acknowledgments ecology: In vitro and in vivo assessments. Food Funct. 7,
The authors gratefully acknowledge the instrumental sup- 3064.
Montgomery, H.A.C., Dymock, J.F., and Thom, N.S. (1962).
port via UHPLC provided by Dr. Amalesh Samanta, Professor
The rapid colorimetric determination of organic ACIDS and
in the Department of Pharmaceutical Technology, Jadavpur
their salts in sewage-sludge liquor. Analyst 87, 949.
University. They also sincerely thank Mr. Bhaskar Das, Re- Moosbrugger, R.E., Wentzel, M.C., Ekama, G.A., and Marais,
search Scholar at the same department for extending help with G.V. (1993). A 5 pH point titration method for determining
regard to analysis of real anaerobic samples via UHPLC. the carbonate and SCFA weak acid/bases in anaerobic sys-
tems. Water Sci. Technol. 28, 237.
Author Disclosure Statement Mueller, H.F., Buswell, A.M., and Larson, T.E. (1956). Chro-
No competing financial interests exist. matographic determination of volatile acids. Sewage Ind.
Waste. 28, 255.
Narkis, N., Henefeld-Fourrier, S., and Rebhun, M. (1980).
References
Volatile organic acids in raw wastewater and in physico-
Albaiges, J., Casado, F., and Ventura, F. (1986). Organic indi- chemical treatment. Water Res. 14, 1215.
cators of groundwater pollution by a sanitary landfill. Water Palacio-Barco, E., Robert-Peillard, F., Boudenne, J.L., and
Res. 20, 1153. Coulomb, B. (2010). On-line analysis of volatile fatty acids in
Anderson, G.K., and Yang, G. (1992). Determination of bicar- anaerobic treatment processes. Anal. Chim. Acta 668, 74.
bonate and total volatile acid concentration in anaerobic di- Peu, P., Béline, F., and Martinez, J. (2004). Volatile fatty acids
gesters using a simple titration. Water Environ. Res. 64, 53. analysis from pig slurry using high-performance liquid chroma-
APHA. (1995). Standard Methods for the Examination of Water tography. Int. J. Environ. Anal. Chem. 84, 1017.
and Wastewater, 19th ed. Washington, DC: APHA. Randall, A.A., Benefield, I.D., Hill, W.E., Nicol, J.P., Boman,
APHA, AWWA and WEF. (1992). Standard Methods for the G.K., and Jing, S.R. (1997). The effect of volatile fatty acids
Examination of Water and Wastewater, 18th ed. Baltimore, on enhanced biological phosphorus removal and population
MD: American Public Health Association. structure in anaerobic/aerobic sequencing batch reactors.
Boe, K., Batstone, D.J., and Angelidaki, I. (2007). An innova- Water Sci. Technol. 35, 153.
tive online VFA monitoring system for the anerobic process, Raposo, F., Borja, R., Cacho, J.A., Mumme, J., Orupõld, K.,
based on headspace gas chromatography. Biotechnol. Bioeng. Esteves, S., Noguerol-Arias, J., Picard, S., Nielfa, A., Schere,
96, 712. P., Wierinck, I., Aymerich, E., Cavinato, C., Rodriguez,
Bulen, W.A., Varner, J.E., and Burrell, R.C. (1952). Separation D.C., Garcı́a-Mancha, N., Lens, P.N.T., and Fernández-
of organic acids from plant tissues. Anal. Chem. 24, 187. Cegrı́, V. (2013). First international comparative study of
Buswell, A.M., Boring, J.R., and Milam, J.R. (1960). A paper volatile fatty acids in aqueous samples by chromatographic
chromatographical method for volatile acids. J. Water Pollut. techniques: Evaluating sources of error. Trends Anal. Chem.
Control Fed. 32, 721. 51, 127.
 VFA MEASUREMENT VIA SPECTROPHOTOMETRIC METHOD 19

Robert-Peillard, F., Palacio-Barco, E., Dudal, Y., Coulomb, B., Wentzel, M.C., Ekama, G.A., and Marais, G. (1991). Kinetics of
and Boudenne, J.L. (2009), Alternative spectrofluorimetric nitrification denitrification biological excess phosphorus re-
determination of short-chain volatile fatty acids in aqueous moval systems—A review. Water Sci. Technol. 23, 555.
samples. Anal. Chem. 81, 3063. Yan, C.T., and Jen, J.F. (1992). Determination of volatile fatty
Siedlecka, E.M., Kumirska, J., Ossowski, T., Glamowski, P., acids in landfill leachates by GC with distillation pretreat-
Gołe˛biowski, M., Gajdus, J., Kaczyński, Z., and Step- ment. Anal. Chim. Acta 259, 259.
nowski, P. (2008). Determination of volatile fatty acids in Yang, M.H., and Choong, Y.M. (2001). A rapid gas chro-
environmental aqueous samples. Pol. J. Environ. Stud. 17, matographic method for direct determination of short-chain
351. (C2-C12) volatile organic acids in food. Food Chem. 75, 101.
Tangerman, A., and Nagengast, F.M. (1996). A gas chromato- Zhang, H., Wang, Z., and Liu, O. (2015). Development and
graphic analysis of fecal short-chain fatty acids, using the validation of a GC–FID method for quantitative analysis of
direct injection method. Anal. Biochem. 236, 1. oleic acid and related fatty acids. J. Pharm. Anal. 5, 223.
Downloaded by University of Adelaide from online.liebertpub.com at 09/08/17. For personal use only.