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Development and validation of a method (GC/TCD) to determine N2, CH4, CO2

and H2S in biogas


Maria Angela Tallarico Adorno*, Carolina A. S. Mirandola, Daniel F. N. Pimenta,
Ins N. Tomita
Laboratory of Anaerobic Processes (LPB), Department of Hydraulics and Sanitation,
EESC/USP So Carlos/SP, Brasil. Av. Joo Dagnone, 1100, Bloco 4F, J. Santa
Angelina, CEP 13563-120
*e-mail: janja@sc.usp.br

Abstract
Anaerobic digestion of organic matter, which is generally used for wastewater
treatment, it occurs the generation of biogas, mainly composed of methane
(CH4) and carbonic gas (CO2), besides occasionally producing sulfidric gas
(H2S), nitrogen (N2) and hydrogen (H2), depending on the composition of the
organic matter present in the wastewater and on the physical-chemical
conditions of the process. Moreover, methane and hydrogen may be used as
alternative sources of renewable energy. So, it is very important to monitor the
biogas (composition and quantification), which is made by gas chromatography.
The validation of the method by gas chromatography with thermal conductivity
detection (GC/TCD) described here was assessed by the following parameters:
linearity (between 0.9948 and 0.9986), precision of the method by the
coefficient of variation (CV %)of the chromatographic areas (from 0.29% to
12.57% related to the chromatographic area) and instrumental precision (from
5.92% to 8.0%) and limits of detection (from 0.06 to 0.20 moles) and
quantification (from 0.19 to 0.60 moles). The results attested that it is suitable
for determination of N2, CH4, CO2 and H2S in the biogas generated by anaerobic
processes treatment of wastewater.
Keywords: anaerobic processes, gas chromatography, composition of biogas.
Introduction
The determination of the composition of the biogas formed in the anaerobic
degradation of organic matter by microorganisms is important because in these
processes are formed mainly carbon dioxide and methane (a renewable energy
source) and, occasionally, H2S, H2, NH3, among others. They can be released to
atmosphere, causing environmental impact and unpleasant smell (3). Anaerobic
digestion is an efficient technology for waste treatment, including municipal,
agricultural and industrial organic wastes. Some advantages of this process are
the low energy consumption and the probability of energy recovery. The
accumulation of organic acids produced in anaerobic digestion, as well as the
decrease of production rate of methane is usually an indicator of the inhibition of
this process (2). The composition of biogas depends on the organic matter
considered, on the pH, alkalinity, temperature and other substances which may
be present in the organic matter to be degraded (5).

Also, the biochemical methane potential test is a parameter used to determine the
methane yield of an organic matter substrate subjected to a specific anaerobic
process and it requires the quantification of the methane in the assay to measure
the digestibility of a particular substrate (4).
In this work we developed a rapid method by GC/TCD (Gas Chromatography
with Thermal Conductivity Detector) for the determination of nitrogen, methane,
carbon dioxide and hydrogen sulfide (N2, CH4, CO2, and H2S respectively) with
manual injection in samples from anaerobic reactors treating wastewater. The
validation parameters were evaluated (linearity, limits of detection and
quantification LOD and LOQ and precision) according to Ribani et al. (2004).
Methods
Two cylinders, one with a standard mixture of N2, CH4 and CO2 and another of
pure H2S were used for the construction of the calibration curves after the
injection of different volumes (from 10 to 1000 L) of the standard gases at
ambient pressure and temperature. The H2S was bubbled into a solution of 6N
NaOH and the injection of all standards was made manually by a plastic syringe
connected to a push-botton valve in which the needle and the syringe are
easily replaceable (Figure 1), through a silicone tube. The injected volumes were
transformed into number of micromoles through Equation 1 and then it was
plotted the number of micromoles against the chromatographic areas. Each
volume of gas was injected by three different persons, in triplicate (9 replicates
at all).
PV = nRT

(Equation 1)

Where: P = ambient pressure (mm Hg); V = injected volume (L); n = number


of micromoles, R = gas constant (62.4 [mmHg. L/moles.K]) and T = ambient
temperature (K).
The chromatographic conditions which provided good separation with well
resolved peaks in a short time were: Shimadzu Chromatograph GC-2014
equipped with a thermal conductivity detector (TCD) and an HP-PLOT/Q (30 m
x 0.53 mm x 40 m) column. Temperature of the injector: 160 C; temperature
of the detector: 170 C; oven temperature: 35 C (2 min) to 170 (1.0 min), at 60
C/min (5.25 min of chromatographic run); split: 5 and column flow: 4 mL min1
. Hydrogen was used as the carrier gas.

Figure 1. Plastic syringe with push-bottom valve

Validation parameters (6)


Linearity was evaluated by means of the coefficient of correlation (R) for the
regression lines and Ftest. The LOD and LOQ were calculated according to
Equations (2) and (3).
LOD = 3.3 * s/S

Equation (2)

LOQ = 10 * s/S

Equation (3)

Where s is the standard deviation of the linear coefficient and S is the average of
the angular coefficient of the equations of the regression lines.
The precision of the method was calculated based on the coefficient of variation
(CV %) of the chromatographic areas (Equation 4) and the instrumental
precision was determined after 10 injections of the same volume of the gases.
CV % = w/ * 100

Equation (4)

Where w is the standard deviation and is the average of the chromatographic


areas.
Results and discussion
The retention times (RT) obtained at the chromatographic conditions described
above for the gases N2, CH4 CO2 were: 1.97, 2.15 and 2.72 (Figure 2),
respectively and for H2S: 4.27 min (Figure 3).
The obtained values for equations of the regression lines, R, Ftest, LOD and
LOQ are in Table 1. The tabulated Ftest values are: 2.72 for the mixture of the
gases (13 levels) and 3.87 for the H2S (8 levels). The values obtained for Ftest
are greater than the tabulated value, which is an evidence of the linearity of the
method (1), in addition to the R values above 0.99 (6).
The method presented low values for LOD and LOQ, besides the low CV % for
the chromatographic areas, which indicates good precision of the method (from
0.29% to 12.57%) for the mixture of gases and (from 0.95% to 7.31%) for H2S
in addition to the low CV % for instrumental precision (Table 2), considering the
manual injection of the gases.

Figure 2. Chromatogram by GC/TCD: 600 L of the mixture of gases

Figure 3. Chromatogram by GC/TCD: 50 L of H2S


Table 1. Equations for the regression lines, R, Ftest, LOD and LOQ
LOD

LOQ

(moles)

(moles)

4555.82

0.20

0.60

0.9985

7134.77

0.06

0.19

y = 19252.23x + 2724.84

0.9986

7884.40

0.09

0.27

y = 27150.47x 5074.59

0.9936

1087.36

0.11

0.33

Gas

Equations

Ftest

N2

y = 18143.03x + 3075.73

0.9976

CH4

y = 14408.18x + 1847.35

CO2
H 2S

Table 2. Parameters evaluated for the determination of the instrumental precision


Chromatographic areas
Gas
Average

Standard
deviation

Retention time (min)


CV %

Average

Standard
deviation

CV %

N2

23426.12

1385.82

5.92

1.942

0.002

0.11

CH4

18131.67

1253.68

6.91

2.141

0.002

0.08

CO2

24582.36

1678.46

6.83

2.741

0.002

0.07

H 2S

95245.96

7617.33

8.00

4.223

0.005

0.11

Conclusion
The validation parameters evaluated attested good linearity, precision and low
limits of detection and quantification to this method, which is suited to
determine N2, CH4, CO2 and H2S in biogas generated in anaerobic processes by
manual injection. Since there are different types of reactors (batch or continuous,
for example) this method allows manual injection with a low cost without the
need for online analysis with special devices for injection.

Acknowledgments
The authors acknowledge financial support from Finaciadora de Estudos e
Projetos (FINEP), Conselho Nacional de Desenvolvimento Cientfico e
Tecnolgico (CNPq) and Fundao de Amparo Pesquisa (FAPESP).
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