PII: S0043-1354(98)00023-2
Wat. Res. Vol. 32, No. 10, pp. 3168±3172, 1998
# 1998 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
0043-1354/98 $19.00 + 0.00
DETERMINATION OF FATTY ACIDS (C8±C22) IN URBAN
WASTEWATER BY GC-MS
A. GONZAÂLEZ CASADO, E. J. ALONSO HERNAÂNDEZ and J. L. VIÂLCHEZ*
Department of Analytical Chemistry, University of Granada, c/ Fuentenueva s/n, E-18071 Granada,
Spain
(First received March 1997; accepted in revised form December 1997)
AbstractÐA simple method for determination of fatty acids (C8±C22) in wastewater, extracted with
dichloromethane, was developed. The extraction time was drastically reduced using ultrasound. Fatty
acids were esteri®ed using BF3 methanolic solution followed by an 11 min run using gas chromatography-mass spectrometry with selected ion monitoring (GC/MS-SIM). A clean-up is not necessary
using SIM Mode. Tridecanoic acid was used as a surrogate internal standard. Detection limits go from
0.008 to 0.016 mg lÿ1. The applicable concentration range was 0.010 to 10 mg lÿ1. The method was validated by using the Standard Addition Methodology and it was applied satisfactorily to wastewater
from Granada (Spain). # 1998 Elsevier Science Ltd. All rights reserved
Key wordsÐwastewater analysis, fatty acid composition, GC±MS, ultrasonic bath, liquid±liquid extraction
INTRODUCTION
Fatty acids have long been associated to cleaning
tasks and because of the nature of such tasks and
of the involved materials (coming from vegetable or
animal sources), the idea that their environmental
harm should not be unreasonable seems to be deeply rooted.
Some doubts however can arise on subjects such
as the potential interactions between Ca/Mg fatty
acid salts and aerobic or anaerobic microorganisms.
For instance, the (Ca + Mg)/Na ratio is substantially higher in anaerobically digested sludges than
in either treated or untreated waters. Organic matter, including soaps, could be precipitated as Ca±
Mg salts in waste water treatment plants
(W.W.T.P.) becoming integrated in the solid/sludge
fractions coming out of the W.W.T.P.s escaping
thus to the biological treatments and/or chemical
processes. This work features our ®ndings on this
subject in the Granada City W.W.T.P.
The analysis of fatty acids from all kinds of
waters (tapwater, seawater and sewage sources) has
usually involved multistep processes i.e. Liquid±
Liquid Extraction or Solid±Liquid extraction and
Chromatography. Pempkowiak (1983) and Sliwiok
and Kozera (1991) used C18 Solid Phase Extraction
in seawater and post-ranation water. Delmas et
al. (1984) and Parrish et al. (1992) used Liquid±
Liquid Extraction after ®ltration followed by thin
layer chromatography in seawater while Wang and
*Author to whom all correspondence should be addressed.
[Fax: +34-58-243328, E-mail: [email protected]].
Zhao (1992) used Multi-Step Liquid±Liquid
Extraction, followed by gas chromatography in
gasi®cation wastewater. In all cases, tedious work
looms ahead if anyone tries to do such things
because low solubility of the fatty acids, Ca, Mg
and K salts associated to particulates of very complex composition and other problems are partially
responsible for it. As quoted by Moreno et al.
(1992), a Soxhlet extraction can take as much as
72 h.
Here, a new method for the analysis of fatty
acids (C8±C22) in wastewater is presented requiring
only 6 h of mechanical stirring in a sonicated bath
of the wastewater acidi®ed suspension followed by
a Liquid±Liquid Extraction.
EXPERIMENTAL
Apparatus and software
All the measurements were performed with a Hewlett
Packard system made up by a 5890 gas chromatograph
®tted with a HP 7673 autosampler, a splitless injector for
capillary columns and a 5971 mss spectrometer with EI
(70 eV) as ionization source and quadrupole mass ®lter. It
was modi®ed to obtain more sensitivity and could be used
in Scan (complete spectrum in a m/z range) and SIM
(selected ion monitoring) modes. The mass spectrometer
was calibrated every day before use with per¯uorotributylamine (PFTBA) as a calibration standard.
The column was a HP1 fused silica capillary
(30 m 0.25 mm i.d., 0.25 mm ®lm thickness) coated with
methyl silicone gum phase. The computer was a HP-UX
Chemsystem with a spectral library of more than 140 000
compounds. It was the controller of all chromatographic
system. Carrier gas was helium (purity 99.999%).
Statgraphics 6.0 (1992) software package was used for the
3168
Urban wastewater analysis
statistical analysis of data. The Lack-of-®t test was applied
to check the linearity of the calibration graphs according
with the Analytical Methods Committee (1994).
Reagents
All the fatty acids, analytical-reagent grade 99%, i.e.
caprylic acid C8, capric acid C10, lauric acid C12, myristic
acid C14, palmitic acid C16, oleic acid C18:1, stearic acid
C18, arachidic acid C20 and behenic acid C22, and the tridecanoic acid C13, used as internal standard (IS), as well
as the 14% boron tri¯uoride (BF3) methanol solution used
for esteri®cation were purchased from SIGMA.
Stock solutions of fatty acids containing 100 mg lÿ1
were prepared in 500 ml volumetric ¯asks, dissolving
50.0 mg of the compound in 96% (v/v) methanol
(Panreac). A standard solution of 100 mg lÿ1 of tridecanoic
acid was used as an surrogate internal standard.
All solutions were stored in dark bottles at 48C, remaining stable for at least six months.
Sample treatment
Water samples were collected in glass bottles previously
cleaned with HCl and stored at 48C until analysis. The
usual precautions were taken to avoid contamination.
Analysis were performed with the least possible delay.
Procedure
Extraction method. 5 ml of wastewater were diluted to
100 ml and HCl (1:1) was added to bring the pH to 1. The
acid suspension was mechanically stirred in a sonicated
bath for 6 h.
Then, the sample was transferred to a separatory funnel
and 5 ml of dichloromethane were added. The mixture
was shaken for 1 min and the organic phase was collected
after decantation. The extraction was repeated with 5 ml
of dichloromethane. The extracts were mixed, dried over
anhydrous sodium sulfate, ®ltered and concentrated to
dryness in a rotary vacuum evaporator. The extract was
3169
dissolved in 3.0 ml of methanol and 1 ml of methanolic
solution of 1 mg lÿ1 tridecanoic acid (IS). The fatty acid
mixture solution was collected in a clean dry test tube
being then ready for the esteri®cation step.
Esteri®cation method. 2.0 ml 14% BF3 methanolic solution were added to the fatty acid mixture methanolic solutions and the whole placed in a 708C water bath for
3 min. Then, 1.0 ml of deionized water was added in order
to stop the reaction by cooling. The fatty acid methyl
esters were extracted from the aqueous methanol phase by
adding 1 ml of methylene chloride and shaking the test
tube for 1 min to favor mixing. Two layers were formed,
the methylene chloride layer was drawn o€ with a Pasteur
pipet and transferred to another test tube while the aqueous methanol phase was extracted twice again with 1.0 ml
of methylene chloride. The extracts were then mixed, dried
over anhydrous sodium sulfate, ®ltered and concentrated
to 1 ml with a nitrogen stream.
Calibration. Five methanolic solutions containing mixtures of (C8±C22) fatty acids of known concentrations, (0,
2.5, 5.0, 7.5, 10.0 mg lÿ1), and 1 mg lÿ1 of the IS solution
were esteri®ed as described in the esteri®cation procedure
and directly injected in the gas-chromatograph. A calibration graph was constructed for each fatty acid.
GC-MS analysis. A two-microliter aliquot of the extract
containing the methyl esters was injected by the autosampler in the injector using splitless mode with the split
closed for 2 min. The gas chromatograph parameters were:
Total Flow 100 ml minÿ1, Septum Purge 3 ml minÿ1, Head
Column Pressure 105 kPa, Injector Temperature 2008C,
oven temperature program: 758C (1 min), 308C/min, 2708C
(7 min). The mass spectrometer parameters were: Interface
Temperature 2808C, Electron Multiplier Voltage between
1750 and 2100 V, Scan mode m/z range 45±500. The
selected ions of the compounds for SIM mode operation
were m/z 55, 74 and 87.
Concentrations of the fatty acids were calculated using
the internal standard method.
Fig. 1. In¯uence of the sonication/stirring time in the chromatographic signal. (1) caprylic acid (2) oleic
acid (3) behenic acid.
A. GonzaÂlez Casado et al.
3170
Fig. 2. Typical chromatogram of wastewater fatty acids analysis of the W.W.T.P. of Granada City
(Spain), obtained in SIM Mode.
RESULTS AND DISCUSSION
Due to the particulate nature of wastewater, we
decided to use a liquid±liquid extraction of the 20:1
diluted samples to be analyzed. We studied the in¯uence of the pH of the medium in the extraction
eciency of the fatty acids. Due to its weakly acid
nature, the eciency of the extraction decreased
slightly when the pH increased till about pH 4±5,
being drastically reduced at higher pH values while
increasing amounts of lather were generated,
namely when the system was shaked. Therefore,
fatty acid extractions were carried out at pH values
around 1 by adding a few drops of a 1:1 hydrochloric acid water solution.
We screened seven di€erent solvents: n-hexane,
iso-octane, ethyl±ether, methylene chloride, trichloromethane, carbon tetrachloride and trichloro±
tri¯uoro±ethane selecting methylene chloride as the
more adequate. To preconcentrate the analytes to
the required ®nal concentration without using too
high amounts of organic solvent, a 20:1 ratio was
found appropriate enough.
The e€ect of mechanical stirring in a sonicated
bath on the fatty acid recovery at pH 1 was monitored through stirring times ranging from 3 to 24 h.
A 6 h duration appeared to be suitable, since the response in the chromatographic signal was maximum
at that time (Fig. 1). The use of a sonicated bath
obviously allowed such a short extraction time.
The ionic strength did not a€ect the extraction
eciency in the above mentioned experimental conditions.
A typical chromatogram obtained in a real
sample with the above described set-up conditions
is shown in Fig. 2. Only 11 min were necessary to
complete an analysis.
In a SIM analysis a high mass number and a
high intensity were chosen in order to obtain good
sensibility and to prevent interferences.
The mass spectrum of the fatty acid methyl-esters
were carried out in Scan Mode. The molecular ions
appear at the corresponding molecular weight. The
base peak corresponds to the McLa€erty rearrangement and appears at m/z 74, except in the case of
oleic methyl ester (base peak, m/z 55). A relevant
peak corresponding to speci®c 8-center rearrangement and H shift appears at m/z 87 in all cases.
Other speci®c peaks showed a lower abundance.
Due to its higher abundance we have selected m/z
74 as a Target ion and m/z 53 and 87 as Quali®er
ions for the SIM mode analysis.
Analytical parameters
Calibration graphs for samples treated according
to the analytical procedure above described were
made using the SIM mode. They are linear for the
concentration range 0.010±10.0 mg lÿ1 of each fatty
acid. In order to check the linearity of the calibration standard the lack-of-®t test Analytical
Methods Committee (1994) was applied for two
replicates and three injections of each standard. The
results for the intercept (a), slope (b), correlation
coecient (r) and probability level of lack-of-®t test
(Plof(%)) are summarized in Table 3. Thus, the data
yield a good linearity within the stated range.
Urban wastewater analysis
3171
Table 1. Analytical parameters
Fatty acids
a
b
0.0004
ÿ0.002
ÿ0.001
ÿ0.001
0.0012
0.0010
0.0023
ÿ0.001
0.0006
C8
C10
C12
C14
C16
C18
C18:1
C20
C22
r
0.899
0.964
0.955
0.926
0.948
0.957
0.896
0.829
0.759
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
0.999
LOF
DL
QL
62
31
87
63
29
58
15
70
16
0.010
0.012
0.010
0.014
0.008
0.012
0.016
0.012
0.013
0.034
0.041
0.032
0.043
0.025
0.039
0.025
0.039
0.052
a, intercept; b, slope; r, correlation coecient; LOF, Lack-of-®t test (p-value) %; DL, detection limit (mg lÿ1); QL, quanti®cation limit
(mg lÿ1).
There is not agreement about how to get the
detection limits (DL) and quanti®cation limits (QL)
from the blank standard deviation in gas chromatography, in contrast with other analytical techniques. Frequently, IUPAC recommendations are
not strictly used.
We believe that the method that we have applied,
GonzaÂlez-Casado et al. (1996), for calculating DL
and QL in pesticides in water is more in line with
the IUPAC recommendations. It relies in studying
the blank standard deviation in an interval of time
corresponding to the peak width in its base, extrapolated to zero concentration.
Here, DL and QL were estimated as in the above
reference. Other analytical parameters summarized
in Table 1 were established by applying the method
proposed by Cuadros-RodrõÂ guez et al. (1993).
Validation and applications of the method
The validation of the proposed method to wastewater samples was carried out by using the
Standard
Addition
Methodology,
CuadrosRodrõÂ guez et al. (1995). Three experiments are
required to obtain the data set necessary to obtain
the proposed statistical protocol. In each one the
same analytical procedure is applied:
(a) standard calibration (SC) as described above.
(b) standard addition calibration (AC): This calibration was obtained by standard additions of fatty
Table 2. Numerical values of parameters SC, AC and YC
Fatty
acids
C8
C10
C12
C14
C16
C18
C18:1
C20
C22
a
SC
AC
YC
SC
AC
YC
SC
AC
YC
SC
AC
YC
SC
AC
YC
SC
AC
YC
SC
AC
YC
SC
AC
YC
SC
AC
YC
0.0004
0.6319
0.0078
ÿ0.021
0.8647
0.0017
0.0097
2.8657
ÿ0.0550
ÿ0.0054
2.1469
ÿ0.0367
ÿ0.0021
4.6369
0.0683
0.0010
0.5735
0.0013
0.0047
6.8211
ÿ0.0583
ÿ0.0014
0.2458
0.0007
0.0006
0.3813
0.0035
b
0.899
0.893
1.249
0.964
0.969
1.7385
0.898
0.906
5.863
0.9305
0.9276
4.3387
0.9425
0.9304
9.0840
0.9574
0.9621
1.1451
0.9175
0.9090
13.2707
0.8295
0.8319
0.4920
0.7585
0.7599
0.7599
Syx
0.0056
0.0081
0.0062
0.0076
0.0117
0.0140
0.0802
0.0421
0.0640
0.0694
0.0669
0.0747
0.0619
0.0437
0.1334
0.0072
0.0101
0.0072
0.0535
0.1018
0.1198
0.0065
0.0078
0.0059
0.0064
0.0061
0.0120
t(b)
bp
a'
cx
t(c)
1.84
p = 71%
0.897
0.003
0.628
0.693
0.691
0.74
p = 46%
0.62
p = 54%
0.966
ÿ0.003
0.866
0.894
0.895
0.08
p = 90%
1.36
p = 18%
0.901
ÿ0.005
2.893
3.259
3.273
0.55
p = 58%
0.48
p = 64%
0.930
ÿ0.001
2.137
2.331
2.339
0.32
p = 75%
1.30
p = 20%
0.938
0.017
4.598
4.839
4.827
0.55
p = 58%
0.7517
p = 46%
0.959
ÿ0.000
0.575
0.598
0.599
0.12
p = 90%
1.38
p = 18%
0.915
0.019
6.791
7.487
7.489
0.04
p = 95%
0.43
p = 66%
0.830
ÿ0.002
0.2467
0.297
0.296
0.14
p = 98%
0.27
p = 78%
0.759
0.000
0.382
0.496
0.498
0.74
p = 54%
Syx: regression standard deviation; t(b): statistic for slope; bp: pooled slope of AC and SC; a': corrected intercept; cx: analyte content; t(c):
statistic for analyte content.
A. GonzaÂlez Casado et al.
3172
Table 3. Fatty acid composition of a sample from Granada wastewater
Fatty acids
Caprylic acid (C8)
Capric acid (C10)
Lauric acid (C12)
Myristic acid (C14)
Palmitic acid (C16)
Oleic acid (C18:1)
Stearic acid (C18)
Arachidic acid (C20)
Behenic acid (C22)
mg lÿ1
%
0.362 0.04
0.432 0.04
1.632 0.04
1.192 0.04
2.412 0.08
0.302 0.04
3.732 0.06
0.162 0.04
0.242 0.04
3.4
4.1
15.6
11.4
23.1
2.9
35.7
1.5
2.3
ÿ1
acids (0.00, 0.25, 0.50, 0.75 and 1.00 mg l ) to
sampled wastewaters, the sampled volumes being
always 100 ml.
(c) Youden calibration (YC): A calibration curve
was made with the Youden method, Cardone
(1986).
Increasing amounts of sample volume (25, 50, 75
and 100 ml respectively) were checked three times
for each of the above mentioned concentrations. By
applying linear regression analysis, the slope, the
intercept, and the regression standard deviation for
each curve representing the whole range of spiking
concentrations are calculated. for each of the three
methods. The parameters obtained from these three
checkings are shown in Table 2. The Student t test
shows the similarity of the representatives values of
slope deduced for the standard calibration (SC) and
standard addition calibration (AC) methods.
Results are not signi®cantly di€erent, and it can
be concluded that our method is accurate. On the
other hand the non-existence of an intercept in the
Youden calibration (YC) implies the absence of
matrix e€ect.
The proposed method was applied to a wastewater of Granada (Spain) where we found indeed
fatty acids in the amounts stated in Table 3.
CONCLUSIONS
A simple and practical method for the analysis of
fatty acids in wastewater by GC-MS is presented.
Extraction time is drastically reduced using ultrasound. Shorter extraction times with the technique
described here should be interesting for people
involved with wastewater disposal monitoring. The
method was applied satisfactorily to wastewater
samples of a W.W.T.P. of Granada City (Spain).
AcknowledgementÐThis research was supported by the
Comision Interministerial de Ciencia y Tecnologia
(CICYT) (Project No. AMB-97-1222).
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