Polish J. of Environ. Stud. Vol. 17, No.

3 (2008), 351-356

Original Research

Determination of Volatile Fatty Acids in

Environmental Aqueous Samples
E.M. Siedlecka, J. Kumirska, T. Ossowski, P. Glamowski, M. Gobiowski,
J. Gajdus, Z. Kaczyski, P. Stepnowski*
Faculty of Chemistry, University of Gdask, ul. Sobieskiego 18, PL-80-952 Gdask

Received: September 12, 2007

Accepted: January 24, 2008
Abstract
Volatile fatty acids (VFAs) are present in environmental waters in the range of 1 to 5,000 ppm and
different methods have been reported for their determination. In this paper we have studied and compared
analytical performance parameters for the distillation method followed by potentiometric titration, spectrophotometric and gas chromatographic methods. The main disadvantage of the distillation approach was
quite poor absolute recovery (53-58%) from the given matrix and rather elevated limit of quantification
(LOQ) at 110 mg/L. Direct potentiometric titration was characterized by acceptable accuracy (above 97%)
and precision in the range 1.8%-15%. The LOQ value was 11 mg/L. The spectrophotometric method was
sensitive for hydrogen carbonate alkalinity and phosphate ions; measured concentrations of acetic acid
were lower than nominal. The precision and accuracy of the spectrophotometric method were in the ranges
1.3-14% and 82.1-104.2%, respectively. Limit of quantification was 28 mg/L. However, if ion exchange
bed is used prior to this method the LOQ can be reduced to 5 mg/L. The GC method is characterized by
quite low LOQ (5 mg/L) and seems to be the best methodology to determine low VFA concentrations in
environmental waters. The precision of the method ranged from 5.7 to 14.8% and accuracy was above 92%.
Additionally, this method allows for determination of individual VFAs.

Keywords: volatile fatty acids, environmental waters, spectrophotometry, gas chromatography, distillation, potentiometric titration

Introduction
Low-molecular mass carboxylic acids, (C2-C7 monocarboxylic aliphatic acids) are important intermediates
and metabolites in biological processes. These carboxylic
acids are known as volatile fatty acids (VFAs) or shortchain fatty acids (SCFAs). The presence of VFAs in a
sample matrix is often indicative of bacterial activity.
VFA analysis is significant in studies of health and disease in the intestinal tract [1]. In some foods VFA content
*e-mail: [email protected]

is an index to quality assurance [2]. Volatile fatty acids

originate from anaerobic biodegradation of organic matter. Therefore, they are widely present in activated sludge
[3], waste and landfill leachates [4], and wastewater. Recently, the determination of VFAs has become of increasing interest since it has been found that they are involved
in different processes, for example in biological removal
of phosphorus from water [5] or nitrification- denitrification in activated sludge [6]. Carboxylic acids may also affect the storage stability of waste incineration residues by
reducing pH value and increasing the mobility of heavy
metals and radionuclides. In addition, VFAs constitute
one of the chemical classes responsible for unpleasant

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Siedlecka E.M. et al.

odour generated in wastewater, together with amines and

sulfur compounds.
Additionally, large quantities of potassium and sodium salts of VFAs are used as airplane, runway and
apron deicers. After being used, these compounds can
contribute to the increase of chemical oxygen demand,
(COD) thus decreasing overall water quality. In winter,
the biodegradation of used deicing solutions can be limited by microbial inhibition due to low temperatures. In
these seasons, VFAs are usually accumulated in various
environmental compartments and their concentrations increase in waters [7].
Volatile fatty acids exist in environmental matrices in
different concentrations. The results of investigations carried out in Polish municipal wastewater plants concerning VFA levels in wastewater in 2004-05 are presented
in Table 1. Additionally, the range of volatile fatty acid
concentrations in surface water [8] and landfill leachates
are presented [9]. It has been noticed that the range of
VFA concentrations is wide; the content may vary from
a few mg/l in surface water to thousands mg/L in landfill
leachates.
In literature different methods for the determination of
VFAs in environmental samples are reported. Traditionally, the VFA content in wastewaters and foods has been
analyzed by titrimetric or gas chromatographic methods
preceded by solvent extraction. The rapid colorimetric
determination of organic acids are also in use [10]. More
recently, ion chromatography and HPLC [11] have also
been applied to VFA analysis.
This paper investigates advantages and disadvantages of simple procedures used for the routine analysis
of volatile fatty acids in wastewater: distillation, spectrophotometric and gas chromatographic method. Our
work aimed primarily to verify the repeatability, precision and limits of quantification of investigated methods and to estimate the matrix influence of the obtained
results.

Materials and Methods

Chemicals and Materials
All reagents were of analytical grade and used without further purification. All reagents were purchased from
PPH POCh SA (Gliwice, Poland), unless otherwise indicated. The inorganic salts: NaH2PO4 and NaHCO3, were
acquired from Chempur (Piekary lskie, Poland).

Determination of VFA by a Distilled Method

500 mL water samples containing various concentrations of acetic acid were steam-distilled in accordance
to the official Polish Normalized Committee methods
PN-75/C-04616 [12] and as described in Standard Methods for Examination of Water and Wastewater (APHA,
AWWA, WEF, Washington 1992). The content of volatile
acids in distillate was determined by titration with 0.1N
NaOH solution and expressed as acetic acid content. This
procedure was modified in this study by replacement of
the conventional titration with the potentiometric titration
system. The concentration of NaOH solution was matched
with acetic acid content in samples every time.

Determination of Acetic Acid by a

Spectrophotometric Method
The spectrophotometric procedure is based on the well
known colorimetric ferric hydroxamate method for determination of carboxylic esters known as the Montgomery
method [10]. In this study the procedure was modified as
follows: aqueous sample (0.5 mL) was taken into a dry
test-tube, and then after the addition of 1.5 mL ethylene
glycol reagent and 0.2 mL of 19.5N sulphuric acid the
mixture was heated for 3 min in a boiling bath. Then, the

Table 1. The range of volatile fatty acid concentrations in wastewater and water.
Sample
Municipal wastewater influent to biological treatment stage a)
Municipal wastewater influent to wastewater plant a)

population equivalent of municipal wastewater treatment plant

Range of VFA concentrations

450,000

65-168 mg CH3COOH/L

27,000

124-150 mg CH3COOH/L

8,000

70-150 mgCH3COOH/L

450,000

28-69 mg CH3COOH/L

Landfills leachate b)

0- 19,000 mgCH3COOH/L

Water from the airport region collected in

winter a)

52-2,500 mg CH3COOH/L

Surface water c)

0-3 mg CH3COOH/L

a VFAs were determined by distilled method (PN-73/C-04616). Investigations were carried out in Poland in 2004-05 by authors (data
unpublished), b [9], c- [8].

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Determination of Volatile...
content of the test-tube was immediately cooled in cold
water. After cooling, 0.5 mL of 10% hydroxylamine hydrochloride solution, 2 mL of 4.5 N NaOH solution and
10 mL of 10% ferric chloride solution were added. The
absorbance was measured at 495 nm by spectrophotometer (Odyssey HACH). The standard solutions of acetic
acid (1200 mg/L CH3COOH) were diluted with deionized
water to obtain appropriate concentrations in the range of
10 to 1200 mg/L. Deionized water was obtained in-house
by treating tap water with a carbon filter, reversed osmosis, a mixed bed of ion exchangers and a 0.45 m filter.
The effect of the alkalinity and phosphate ions on the
spectrophotometric response for the acetic acid was also
investigated.
The application of ion exchanger (Amberlite IRA 410)
for VFA separation has been also investigated. The different volume of feed solution was subjected to the top of the
column and flow down. VFAs were eluted using different
volume of saturated solution of NaCl at the flow rate 0.4
mL/min. The analytes were collected and determined by
spectrophotometric method.

Determination of VFAs by Gas Chromatographic

Method
The preparation of samples for determination of VFAs
by GC method was based on Manni and Carons procedure [4]. Briefly, the samples were acidified to pH 2 using 65% of nitric acid.1 mL portions were shaken along
with 1 mL of diethyl ether for approximately 10 min, and
the ether phases were quantitatively transferred to 4-mL
flasks, where a small amount of anhydrous sodium sulfate was added. The 500 L portions of ether phases were
transferred into new 4-mL flasks and 150 L of diazomethane was added.
A series of VFA standards for the calibration curves
were prepared in the same manner as described above.
Calibration curves were obtained using five aqueous solutions of acids: acetic, propionic, butyric, valeric, and caproic, in the concentration range of 5 to 1000 mg/mL.
GC analyses were performed on a GC 8000 TOP (CE
Instruments) gas chromatograph equipped with a flame
ionization detector and a DB-23 capillary column (30 m,
0.25 mm I.D., 0.25 m film thickness, Alltech, Poland).
The injector and detector temperatures were both 170C.
The carrier gas was argon. The analyses were performed
using a temperature programme: 5 min at 30C and a linear
gradient from 30C to 130C at 10C min-1. In each case a
2 L of sample was injected (a flow splitting 1:10).

from the matrix. In the distillation method, at first the

wastewater sample is steam-distilled. Then, collected distillate of carboxylic acids is titrated with standard alkaline
solution.
In our study validation parameters of distilled method
was investigated and the obtained results are presented in
Table 2.
The precision of the method was determined by calculating the relative standard deviation (RSD) while accuracy was evaluated through the comparison between
the measured and nominal concentrations of acetic acid in
measured samples (%). The results clearly show that mean
values of measured concentrations (AVG values) were significantly lower than nominal concentrations. Therefore,
the validation parameters of potentiometic titration itself
were also investigated. Good results were obtained in the
range from 11 to 1164 mg/L of acetic acid. The potentiometric method was characterized by acceptable accuracy
(above 97%) and precision in the range 1.8%-15%, (Table
3). Limit of quantitation (LOQ) was 11 mg/L.
Secondly, the absolute recovery of acetic acid from
the matrix was studied. The results given in Figure 1 indicate that the absolute recovery of acetic acid in the range
from 11 to 1156 mg/L was quite poor; only 53%-58% of
nominal concentration was found in a distillate.
Table 2.
Assay validation results for acetic acid analysed by distillation method. Measurements of acetic acid concentrations
made through automated potentiometric titration with 0.1N
NaOH solution (n = 4).
Acetic acid
Nominal concentration [mg/L]

110

123

978

1200

AVG [mg/L]

64

70

567

636

SD

1.5

1.3

14.6

31.1

Precision (RSD%)

2.3

1.8

2.6

4.9

Accuracy (%)

58.2

56.9

58.0

53

AVG-
mean value of measured concentration
, SD-
standard deviation, RSD- relative standard deviation

Table 3.
Assay validation results for acetic acid analyzed by automated potentiometric titration with
0.1N NaOH solution
without distillation step (n = 4).
Acetic acid
Nominal concentration
[mg/L]

11

110

123

978

1200

Results and Discussion

AVG [mg/L]

12

111

124

972

1164

Determination of VFAs by Distillation Method

SD

1.8

2.6

2.2

25

57

Precision (RSD%)

15

2.3

1.8

2.5

4.9

99.3

97.0

The routine determination of volatile fatty acids in

wastewater is preceded by quantitative isolation of VFAs

Accuracy (%)

109.0 100.9 100.8

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Siedlecka E.M. et al.

Fig. 1. Absolute recovery of acetic acid in distillate. Acetic acid

concentrations measured with automated potentiometric titration with 0.1N NaOH solution.

It must also be noticed that acetic acid, which does not

form an azeotropic mixture with water, was particularly
difficult to recover. Therefore, accurate analysis of acetic
acid by distillation method in wastewater is mainly dependent on recovery from matrix.

Spectrophotometric Determination of VFAs

The spectrophotometric determination of VFAs is designed specifically for determining volatile fatty acids in the
digester sludge. The method is based on estrification of the
carboxylic acids present in sample and subsequent determination of the esters by the ferric hydroxamate reaction.
All volatile fatty acids present in the sample are reported as
their equivalent mg/L of acetic acid. This spectrophotometric method is characterized by a relatively simple procedure,
commonly available reagents and quite short time of analysis. The validation results obtained for spectrophotometric
determination of acetic acid are shown in Table 4.
The precision and accuracy of the method was in the
range 1.3-14% and 82.1-104.2% for concentrations of ace-

tic acid between 28-450 mg/L, respectively. Limit of quantification (LOQ) was 28 mg/L. A single analysis by this
procedure takes 25 min., thus it is suitable for multiple determination procedure. Other advantages of this methodology are that the volume of sample is quite small (0.5 mL)
and the determination is carried directly from the sample.
Separation of VFAs from the matrix by ion exchange
bed was also investigated. The optimal conditions of ion
exchanger performance for 300 mL of the feed solution
were 3 mL/min. flow rate and 10 mL of eluent (saturated solution of NaCl). The ion exchanger bed used allowed us to reduce the limit of quantification to 5 mg/L
of CH3COOH.
The effect of alkalinity and phosphate ions (a common
measure of the buffer capacity of environmental waters)
on the spectrophotometer response for VFA determination
was also investigated. The results are shown in Figure 2.
It is clear that in the presence of hydrogen carbonate and
phosphate ions, measured concentrations of acetic acid
were lower than nominal.

Determination of VFAs by Gas Chromatography

Determination of VFAs by GC method included few
important steps: acidification of sample by nitric acid, extraction of VFAs to diethyl ether phase, conversion of VFAs
into methyl esters and final chromatographic analysis of
obtained derivatives. For quantitative analysis the external
standard methodology was used. Quantitative calibration
proceeded for five acids: acetic, propionic, butyric, valeric,
and caproic, in concentrations ranging from 5 to 1000 mg/
mL. A series of VFA calibration standards were prepared in
the same manner as for samples. A GC chromatogram of
results obtained for the standard solutions of VFAs methyl
esters (500 mg/mL in water) is presented in Fig. 3.

Table 4. Assay validation results for acetic acid analyzed by

the spectrophotometric method based on the determination of
carboxylic acid esters measurement (495 nm) according to the
Montgomery method [10] (n = 4).
Acetic acid
Nominal concentration
[mg/L]

28

70

138

198

309

450

AVG [mg/L]

23

73

132

192

313

457

SD

3.1

3.6

3.2

2.6

6.4

43.6

Precision (RSD%)

14

4.9

1.7

1.3

2.0

9.5

Accuracy (%)

82.1 104.2 95.6 96.9 97.0 97.0

Fig. 2. Influence of hydrocarbonate (2 mval/L) and phosphate

ions (1.1 mg/L PO43-) on the determination of acetic acid with
the spectrophotometric method based on the determination of
carboxylic acid esters measurement (495 nm) according to the
Montgomery method [10]

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Determination of Volatile...

Table 5. Assay validation results for acetic acid analysed by GC

method. DB-23 capillary column (30 m, 0.25 mm I.D., 0.25 m
film thickness, Alltech, Poland).
The injector and detector temperatures at 170C. The carrier gas was argon. The temperature
programme: 5 min at 30 C and a linear gradient from 30 C to
130 C at 10 C min-1. In each case a 2 L of sample was injected
(a flow splitting 1:10).
Acetic acid

Fig. 3. GC chromatogram of methyl esters obtained from standard solutions of five acids (each 500 mg/mL): acetic, propionic,
butyric, valeric, and caproic acids. The preparation of samples
for there determination by GC method was based on Manni
and Carons procedure [4]. Chromatographic conditions as follows: DB-23 capillary column (30 m, 0.25 mm I.D., 0.25 m
film thickness, Alltech, Poland). The injector and detector temperatures at 170(C. The carrier gas was argon. The temperature
programme: 5 min at 30C and a linear gradient from 30C to
130C at 10C min-1. In each case a 2 L of sample was injected
(a flow splitting 1:10).

The calibration curve of the most important VFA acetic acid was linear over the range from 5 to 1000 mg/L
with the square correlation coefficient of 0.9994, which
confirmed a good fit to the regression lines (Fig. 4).
Precision and accuracy of the method was determined
by analysis of replicates (n = 6) of samples containing
known concentrations of acetic acid. RSDs ranged from
5.7 to 14.8% and accuracy was above 92% (Table 5).
These results demonstrated acceptable accuracy and precision of the presented method. The limit of quantification

Fig. 4. Curve calibration of acetic acid obtained by GC method,

where: y peak-area of methyl derivative of acetic acid; x the
concentration of acetic acid in water. DB-23 capillary column
(30 m, 0.25 mm I.D., 0.25 m film thickness, Alltech, Poland).
The injector and detector temperatures at 170(C. The carrier gas
was argon. The temperature programme: 5 min at 30C and a
linear gradient from 30C to 130C at 10C min-1. In each case a
2 L of sample was injected (a flow splitting 1:10).

Nominal concentration
[mg/L]

50

AVG [mg/L]

4.6

49.2

111.5 484.6 1000.7

SD

0.68

4.82

8.03

32.95

57.04

Precision (RSD%)

14.8

9.8

7.2

6.8

5.7

Accuracy (%)

92

98.5

111.4

96.9

100.7

100

500

1000

for acetic acid was 5 mg/mL and it was the lowest standard concentration in the calibration curve.
Developed GC method could be a reliable alternative
to other analytical procedures for the analysis of VFAs. A
significantly lower limit of quantification was achieved. The
sample preparation procedure is rather simple and demands
only 2 mL of the aqueous sample. In addition, this method allows for the measurement of individual VFA concentration.
In the case of the presence of additional volatile contaminations in the analyzed water sample, selectivity problems with
determination of acetic acid can appear. The main disadvantage of this method is necessity to prepare a derivatives
agent, diazomethane, which is toxic and non-stable, on the
other hand allowing an increase analytical sensitivity.

Application of Examined Methods to

Environmental Samples
The studied methods of VFA determination were used
for the measurement of VFA concentrations in exemplary
surface- and wastewater samples. Both type of samples
were collected from a Polish airport facility in winter
2006. Each winter, sodium and potassium acetates are
used there as airplane runway and apron deicers. In this
season, low temperatures usually inhibit biodegradation,
therefore VFAs may be present not only in surface- but
also in wastewaters.
In the case of GC method, the concentrations of VFAs
were calculated as a sum of concentrations of acetic, propionic, butyric, valeric and caproic acids The results are
presented in Table 6.
The agreement between investigated methods is not sufficient. Results obtained by distillation method were lower
than those obtained by spectrophotometric procedure. This is
primarily due to a very low absolute recovery of acetic acid
by steam distillation. Differences in results are also due to the
presence of inorganic ions (HCO3-, PO43-) originated from the
matrix. The GC method seems to be the most reliable between
all studied methods determining low VFA concentrations.

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Siedlecka E.M. et al.

Table 6. VFAs determined in environmental samples by distilled, spectrophotometric and GC methods.

Sample

Wastewater

Surface
water from
the airport

Concentration of VFAs
expressed as mg/L of CH3COOH
Distillation SpectrophotoGC method
method
metric method
84

128

124

146

n.d.

98

120

1630

1920

410

560

n.d.

n.d.

27

n.d.

n.d.

22

n.d.

n.d.

15

n.d.

n.d.

18

n.d.

n.d.

13

n.d.

n.d.

22

n.d. not detected (below LOQ) not analyzed by this method

Conclusions
In conclusion, we have examined in detail the most
popular analytical procedures applied for determination
of VFAs in environmental waters. It was found that the
distillation method commonly used in routine tests suffers
from relatively high limit of quantification and very low
precision. These parameters are far more improved if the
spectrophotometric method is used. Moreover, relatively
short time of analysis allows to propose this method for
high throughput measurements, usually demanded in environmental analysis. Additionally, a pre-separation step
onto ion exchanger bed significantly reduces the limit of
quantification. Determination of VFAs by gas chromatographic method is characterized by satisfactory precision
and good accuracy. The limit of quantification is sufficient
for typical environmental concentrations The sample
preparation procedure is rather simple and demands small
volume of the aqueous sample. Moreover, this is the only
method allowing measurement of individual VFA concentrations. Comparison of practical applicability onto exemplary surface- and wastewater samples shows that GC
methods seem to be the most reliable between all studied
methods for determining low VFA concentrations.

Acknowledgements
Financial support was provided by the Polish Ministry of Research and Higher Education under grants DS
8270-4-0093-8 and 8200-4-0085-8.

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