2010 International Conference on Chemistry and Chemical Engineering (ICCCE 2010)

Studies on development of headspace - gas chromatographic determination of

volatile fatty acids in troublesome environmental samples

Bogdan Zygmunt Anna Banel

Gdansk University of Technology Gdansk University of Technology
Gdansk, Poland Gdansk, Poland
e-mail: bogdan.zygmunt@pg.gda.pl

Monika Felchner-wireo
Karlsruhe Institut fuer Technologie
Marta Wasielewska Institut für Ingenieurbiologie und Biotechnologie des
Gdansk University of Technology Abwassers
Gdansk, Poland 76131 Karlsruhe, Germany

Abstract-- A research was done to develop procedure of
determination of volatile fatty acids (VFAs) in heavily polluted
wastewaters using an automated static headspace (HS) unit
combined with gas chromatograph (GC) with flame ionization
detector (FID). Separation conditions were determined under
which VFAs with 2-8 carbon atoms in a molecule separate well
on a Stabilwax capillary column. Factors such as sample
volume, and time and temperature of sample- HS conditioning
have been selected for acetic, propionic, iso- and n-butyric, iso-
and n-valeric and n-capronic acids in the concentration range
0.1- 1000 mg/L. HS conditions were: maintaining the system at
95oC for 30 min, 2mL sample volume placed in 15 mL
extraction vial, acidified to pH 2 and salted out with 0.75 g
NaCl. In quantitation better results were obtained when was
calibration curve divided to the part close to detection limit
and to the part for higher concentrations. Validation process
has been started.
Keywords: volatile fatty acids, headspace, gas
chromatography, trouble some aqueous samples
I. INTRODUCTION
Inflow of polluted media with large content of biogenic
substances to surface waters causes unwanted eutrophication
of these water bodies. Removal of phosphorous and nitrogen
compounds, the most important biogens, from wastewater is
generally based on its biological treatment. In the process of
anaerobic biodegradation of large organic molecules as fats,
carbohydrates and proteins volatile fatty acids (VFAs) are
formed which are a source of easily assimilable organic
carbon for microorganisms and increase the efficiency of
biogens removal. VFAs are also intermediates in conversion
of organic waste into methane, which is increasingly
important due to European and national policies on energy
production, agriculture and the environment [1, 2].
Anaerobic digestion of organic waste in the absence of
oxygen and at neutral pH (optimal pH: 6.7-7.4) gives biogas,
consisting mainly of methane, carbon dioxide and hydrogen
sulphide [3]. This is a complex four step process with the
contribution of microorganisms, involving: hydrolysis,
acidogenesis, acetogenesis and methanogenesis. Each step is
performed by different microorganisms; products of one step
are substrates for the successive step (Figure 1). Hydrolysis
is the step, where biopolymers such as lipids, proteins and
carbohydrates are degraded into higher fatty acids, glycerol,
monomeric carbohydrates, aminoacids and alcohols. The
conversion is possible thanks to extracellular enzymes: lipase,
protease, amylase and cellulose. In acidogenesis, also known
as fermentation, hydrolysis products are converted into
primary volatile fatty acids (VFA), mainly into propionic
acid [4].
In acetogenesis acetogenic bacteria produce acetic acid
from previously obtained VFAs, and homoacetogenic
bacteria use hydrogen an d carbon dioxide for acetic acid
formation [5]. Methanogenesis is the final step of anaerobic
digestion, where microorganisms convert either acetic acid
into methane or to carbon dioxide and hydrogen. Methane
production can also occur through the digestion of
compounds containing one carbon atom, such as i.e.
methanol.
Organic Matter
Fats, carbohydrates, proteins
Hydrolysis
Soluble Organic Molecules
Sugars, amino acids, fatty acids
Fermentation
Volatile Fatty Acids
Acetogenesis H2, CO2
Acetic Acid
Methano-
genesis
CH4 + CO2

978-1-4244-7766-1/$26.00 C 2010 IEEE 281

 2010 International Conference on Chemistry and Chemical Engineering (ICCCE 2010)
Figure 1. Steps of anaerobic biodegradation
VFAs produced in the second step of anaerobic biowaste
digestion – acidogenesis – are low molecular weight
aliphatic monocarboxylic acids with a chain of 2 to 5 [6, 7]
or 6 [8, 9] carbons. Not only are they considered a product of
bacterial activity, but also an important carbon source for
microorganisms and, hence, are crucial in monitoring the
performance of anaerobic digestion [10]. If the rate of their
production exceeds the degradation rate, VFAs accumulation
is observed - especially acetic and propionic acid - which can
result in failure of the process, or even its inhibition (due to
low pH) [10-12].
To study the processes of biological wastewater
treatment and processes of methane production from organic
waste the procedure to determine individual acids in quite
complex matrices is needed. Most often gas chromatography
is a method of choice. However, majority of samples of
interest must be treated in some way before introduction to a
gas chromatograph. Otherwise, poor chromatograms,
technical problems or even column damage can be observed.
The convenient technique to prepare the trouble some
aqueous samples for GC analysis is headspace (HS) whereby
gas phase over the analyzed aqueous sample being at
equilibrium with the sample itself is injected into GC for
analysis. The initial content of a given analyte in the sample
can be derived from its content in HS. The system of HS –
GC can easily be automated allowing many analyses to be
made without human intervention. The technique was
successfully applied to determine a wide spectrum of volatile
organics in polluted waters [13, 14]. Attempts have also been
made to apply the approach to determine alkano
monocarboksylic acids with 2 to 6 carbon atoms in a
molecule in some polluted aqueous samples. The matrices
analyzed included bacterial cultures and wastewaters.
Cruwys e.g. optimized the HS-GC-FID procedure to
determine VFAs in wastewater in a routine way [7]. His
work was aimed at studying sample carry-over and
developing a way of calibration in routine high-throughput
analysis of wastewater. Very often solid phase
microextraction was used to sample volatile components of
an aqueous sample and then thermally desorbed them in a
GC injector.
The aim of this work was to develop an analytical
procedure of VFAs determination in different polluted
media, including leachates from organic waste piles in
ZOO (a) and also samples form full scale and lab scale
digesters (b), using an automated HS-GC equipped
with flame ionization detector (medium a) and GC
coupled with mass spectrometer (medium b). The main
stress was put on the first step of analysis, i.e.
introduction of HS of an aqueous sample to a gas
chromatograph.
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II. MATERIALS AND METHODS
A. Chemicals and standard solutions and samples
Volatile acid standards with 2-6 carbon atoms (C2-C6
acids) of purity of either 99.8% or 99.5% were purchased
from Fluka (Switzerland). Methyl-tert-butyl ether (MTBE)
was from Merck (Germany). Stock solutions of each analyte
in MTBE were prepared by dissolving an acid standard in
MTBE. To optimize GC separation conditions and to
determine retention parameters for the purpose of
identification, water was spiked with a single acid standard
solution (separate aqueous sample for each VFA) in MTBE
at a concentration of 100 g/L and analysed by means of GC-
MS. By mixing the stock solutions and diluting the mixture
to a required concentration a standard solution was produced.
Standard aqueous samples containing each VFA at a
concentration of 500 mg/L were made by spiking purified
water with the standard solution. 2-Ethylbutyric acid solution
was also added to each aqueous sample before analysis.
B. Instrumentation
Headspace gas chromatographic analysis was performed
using a HS XL automatic headspace sampler connected to an
Autosystem XL GC system, both from Perkin-Elmer. 15 mL
standard glass vials, PTBE septa and patented closures were
also from Perkin-Elmer. The temperatures of the HS XL
oven, needle and transfer line were set at 95, 105 and 135 0C,
respectively. The optimized vial thermostating time of the
HS XL unit was found to be 30 min, vial pressurization time
3 min and sample injection time 0.25 min. Temperature of
GC injection port operated in a split mode (1:6) was set to
200 0C. A Stabilwax-DA fused- silica capillary column (30
m x 0.25 mm I.D., film thickness 0.25 m) from Restek was
used for separation. The oven temperature program was:
700C (1min) to 1600C (1min) at 250C/min rate, and then to
2300C (5min) at 100C/min. The total chromatographic run
time was about 18 min. Flame ionization detector
temperature was at 230 0C. To study propionic acid
degradation by microorganisms the applicability of a coupled
HS-GC-MS system was tested.
C. Procedure
2 mL purified water was poured out into a 15 mL
HS vial, and then a standard solution and 2-
ethylbutyric acid solution were added. The vial was
closed with a silicone membrane and an aluminum cap,
shaken vigorously and placed in the carousel of a HS
device. Injecting HS aliquots into GC started after
conditioning the sample for 30 min at 95°C.
Each sample analysis was followed by two blank runs
(8 mL ultrapure water each time) to protect the system
from the possible carry-over between runs.
 2010 International Conference on Chemistry and Chemical Engineering (ICCCE 2010)
III. RESULTS AND DISCUSSION
A. Gas chromatographic separation and headspace
sample introduction
Chromatographic separation
To achieve chromatograms characterized by symmetrical
and well separated peaks column temperature program
parameters were changed and chromatograms evaluated with
respect to resolution and degree of peak symmetry. The
exemplary chromatogram for the parameters selected is
given in Figure 2.
[a.u.
] Pressure adjustment time.
retention time [min]
Figure 2. HS-GC-FID chromatogram of an aqueous standard sample
containing 9 VFAs and IS (each at concentration 500mg/L): 1-acetic acid,
2- propionic acid, 3- isobutyric acid, 4-butyric acid, 5-isovaleric acid, 6-
valeric acid; 7- 2-etylobutyric acid, 8-caproic acid, 9-octanoic acid, 10-
caprylic acid.
Headspace extraction and injection
The HS process was optimized to obtain possibly high
sensitivity and repeatability, while maintaining a reasonably
short analysis time and little labor.
Sample volume.
The volume ratio of two phases, i.e. sample to head space
in a given vial determines a fraction of the analyte extracted
to HS and hence sensitivity of the procedure. HS volume is
also important since it determines maximum volume per
injection for a given number of replicate runs. The geometry
of a vial and the parameters described above affect the time
of reaching equilibrium. So responses (peak areas) and
repeatability (dispersion of replicate measurements) for
different volumes of a standard sample were measured – a 2
mL sample was selected as an analytical procedure
parameter.
Equilibration time.
The head space of a sample introduced into GC can by
used for quantitative measurement provided that equilibrium
between two phases is achieved. The HS of the standard
aqueous sample was injected into GC column applying
283
different conditioning times - 30 min was sufficient time to
achieve equilibrium.
Extraction temperature.
Temperature of headspace extraction is a very important
parameter. The partition coefficient of a volatile analyte
between liquid and gas phase decreases rapidly with
temperature increase; so yield of extraction and hence the
sensitivity of determination should increase with temperature
rise. However, water content in headspace increases, as well
which can have detrimental effect on a chromatographic
system. As a result of the investigations 950C was selected
as a temperature of extraction.
Before the headspace is introduced into GC, the pressure
in a sample vial is increased to a value of carrier gas pressure
at inlet to a chromatographic column. The best repeatability
was found to be for 3 min. and this time was selected.
Volume of head space aliquot injected into GC.
Increasing the volume injected should increase the
response (peak area) and hence the sensitivity of the
procedure. However, if the volume is too high chromatogram
quality can drop and uncertainty of the results increase. In
the studied volume range, i.e. 0.07 – 0.5 mL the differences
in peak areas were not significant. Therefore, 0.5 mL HS was
chosen to be injected.
Salt addition.
Addition of salt to an aqueous solution decreases
solubility of organic compounds in water and in this way
increases the fraction of the analytes extracted to headspace
and hence sensitivity. As found out in earlier studies, 0.375g
NaCl/mL sample was sufficient [15]. When a sample was 8
mL in volume then 3g NaCl was added.
Method validation
The dependence of system response on concentration
was measured in the range of 0.1 – 1000 mg/L of individual
acids in standard aqueous samples. Concentration range was
divided into two parts; from 0 to 25 and from 25 to 1000
mg/L; this increased, i.e. improved the regression coefficient
which is generally lower (worse fitting) at lower
concentrations. This is not suppressing since generally the
response-analyte amount relationship for a concentration
level close to LOD is generally different from that at higher
concentrations. The average error committed in the
headspace analysis described by the coefficient of variation
ranges from 0.12 to 18%. The other validation parameters
are in the process of determination.
IV. CONCLUSION
The research shows that volatile fatty acids containing
from 2 to 6 carbon atoms can be determined with the use of
gas chromatograph (GC) directly connected with automated
headspace (HS) unit. For the 15 mL HS vials applied the best
 2010 International Conference on Chemistry and Chemical Engineering (ICCCE 2010)
results were obtained for an aqueous sample volume of 2
mL. Relatively high temperature of 95oC of head space
process can be applied which assures good extraction yield
even for polar alkanomonocarboxylic acids. The relationship
between system response and analyte concentration is a bit
different for a concentration range close to detection limits
from that for higher concentrations. In quantitation it is better
to apply two different linear equations.
ACKNOWLEDGMENT
The research financed by the Polish Ministry of Science
and Higher Education, grant no. N N523 230535. This
research work was supported by the European Union in the
framework of the European Social Fund. The system project
of the Pomorskie Voivodeship "InnoDoktorant -
Scholarships for PhD students, II edition
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