Determination of Ethanol in Fermented Broth by Headspace Gas

Chromatography using Capillary Column

ASEM HASSAN MOHAMMED 1 , ALAA KAREEM MOHAMMED2 , FIRAS HASHIM KAMAR1 , ASEEL ABDULQADER ABBAS2 , GHEORGHE NECHIFOR3 *
1
Institute of Te chnology- Baghdad, Middle Te chnical Unive rsity, Iraq
2
Departm ent of Biochem ical Engineering, AL-Khwarizm i Engineering College, University of Baghdad, Iraq
3
Departm ent of Analytical Chem istry and Environm ental Engineering, University Politehnica of Bucharest, 1-7 Gheorghe Polizu Str.,
011061, Bucharest, Rom ania

The gas chrom atography (GC) m e thod in analytical che m istry is a quick and accurate m e thod to de te ct
volatile com pone nts like e thanol. A m e thod for de te rm ining volatile com pone nts known as He adspace
chrom atography (HS-GC) was de ve lope d along with an inte rnal standard m e thod (ISM) to ide ntify e thanol
in fe rm e nte d broth in the laboratory. The aim of this re se arch is de te rm ining the conce ntration of e thanol in
fe rm e nte d broth using capillary colum n (ZB-1). This m e thod can analyze e thanol conce ntrations in the
fe rm e nte d m e dium broth ranging from 10 to 200 g/L. The validation of this m e thod was done in orde r to
obtain the re sults to be of high pre cision and the significant, pre cision was re pre se nte d as the re lative
standard de viation (RSD) which was le ss than 5%, accuracy was le ss than 4 % and significance le ve l was
p < 0.05. It was found that this m e thod exhibite d good re producibility.

Ke yw ord s: Fe rm e nta tion, vola tile com pone nts, inte rna l sta nd a rd m e thod , he a d spa ce sa m pling-ga s
chrom atography

Bio-analysis knows a continuous and rapid developm ent
including e le ctroche m ical and, optical m e thods, but was
m a k in g p o s s ib le w ith a d va n c e d c h ro m a to gra p h y
te chnique s [1-4]. Thus, static he adspace gas chrom ato-
graphy (SH-GC) analysis is a proce dure applie d to the
analysis and conce ntration of volatile organic com pounds.
This proce dure is ra the r sim ple a nd to be a ble to ge t
se nsitivity like dynam ic purge and trap ope ration [5, 6].
Laboratory use of this m e thod has incre ase d and has
worldwide validate d to analyze alcohol and blood alcohol
in biological sam ple s and solve nts use d in pharm ace utical
industries. Other known applications of this m ethod include
laboratory analysis of m onom e rs in polym e rs, plastics and
flavor enhancers in beverages, products of food and arom a
in pe rfum e s and cosm e tics [1, 6, 7].
Com plicated sam ples such as biological solution, blood,
cosm e tics a nd pla stics conta in high-m ole cula r w e ight
m a te ria ls, som e of w hich a re non-vola tile tha t ca n be
de posite d into the GC inje ction port a nd le a d to poor
a na lytica l re sult the re fore , lots of a na lysts use se ve ra l
m e thods in laboratorie s for inte nsive sam ple pre paration
to conce ntrate and e xtract inte re st com pounds from this
unde sire d m ate rial. The se e vaporation and conce ntration
proce ss can take a long tim e and m ore cost. Using analysis
of static he adspace will re duce tim e and cost by sam pling
the volatile com pone nt of he adspace dire ctly into the GC
injection port [8, 9]. A headspace sam ple is usually prepared
in a vial which divide in two phase s, liquid phase contain
the vola tile com pone nt, the d ilution solve nt, a m a trix
m odifie r, and vapor phase (he adspace ) at the top of the
vial, se e figure 1. The volatile com pone nts of the m ixture
of com ple x sam ple s (L) will be se parate d from the non-
volatile sam ple com pone nts and colle cte d in the he ad
vacuum or steam part of the sam ple vial (G). An appropriate
volum e of the vapor in the he adspace is w ithdraw n to
deliver to a GC system for partition and detection of volatile
com pone nts [10, 11].
Th e a n a lysis b y u sin g o f ga s c h ro m a to gra p h y c a n
de te rm ine qualitative analysis according to re te ntion tim e
and quantitative analysis using the are a unde r the curve of
a c h ro m a to gra p h ic p e a k w h ic h give s it th e b e s t
pe rform ance com pare with am ong old m e thods. In this
m e thod the sam ple will be store d in a shut vial, the volatile
a na lyte s e va pora te tow a rd the via l he a dspa ce , in this
m e thod of analysis the sam ple is put in a locke d vial. The
volatile com ponents evaporate into the vial headspace was
shown in figure 1. Whe n e quilibrium state be twe e n the
inte re sting volatile analyte s conce ntration in the liquid
p h a se ( sa m p le ) a n d in va p o r p h a se ( h e a d sp a c e ) is
re a c h e d , a n a p p ro p ria te vo lu m e is ta ke n fro m th e
he adspace and inje cte d into the gas chrom atography; this
injection can be carried out m anual or with an auto sam pler
a t h igh e r p re s s u re a n d te m p e ra tu re th a n a m b ie n t
c o n d itio n s [ 12] . No w a d a ys , th e h e a d s p a c e ga s
c h ro m a to gra p h y m e th o d is d e s ire d b e c a u s e th e
contam inants that will re ach to the inje ction port and the
se paration colum n of the gas chrom atography will be as
low as possible [13, 14].
Fig. 1. Phase s of sam ple in the
he adspace containe r, G- the vapor
phase (he adspace ), L- the liquid
phase [10]
Expe rime ntal part
Mate rials and m e thods
Che m icals use d in this re se arch are : absolute e thanol
(HAYMAN, UK); n-butanol (sigm a Aldrich), bake r’s ye ast
(local m arke t), Glucose and othe r che m icals use d in the
fe rm e ntation we re from Sigm a Aldrich.

* em ail: doru.nechifor@yahoo.com

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Sam ple pre paration
Fe rm e ntation m e dium (broth) was pre pare d by m ixing
100 g/L glucose , 8.5 g/L ye ast e xtract, 1.3 g/L NH4Cl, 0.12
g/L MgSO 4.7H2O and 0.06 g/L CaCl2, [15]. Broth m e dium
was inoculate d via bake r’s ye ast by 100 m L of inoculate d
b ro th s a m p le w h ic h w a s p re p a re d in fo rm e r s te p ,
fe rm e ntation proce ss was conducte d in 1 L ste rile shake
flasks, m e tal caps as closure s, and ste rile pipe ts, A shake r
flask incubate at 35 ± 2o C and 150 rpm for about 3 days,
and the n it was coole d to 0 o C and store d for anothe r days.
It w a s ta ke n 5 m L of fe rm e nte d broth sa m ple s a t
d iffe re n t in te rva l tim e s (6, 12, 24, 48, 72 h ) th e n th e
sam ples are taken to centrifuge within one hour after taking
the sam pling. This can be pe rform e d by rotating at 3000
RPM for te n m inute s, and the n plugge d im m e diate ly with
its ground glass stoppe r. Filtrate d portion of liquid of the
sam ple will pass through a 0.45 m m m icro filte r afte r that
, sa m p le a n a lyzin g w a s p e rfo rm a s fo llo w : 2 m L o f
fe rm e nte d broth was m ixe d with 3m l of inte rnal standard
(n-butanol, 4 g/L) in a 10 m L vial, and the n the vial was
tightly close d im m e d ia te ly w ith a suita ble ca ps. Ea ch
analysis was unde rtake n in triplicate with diffe re nt vials.
In orde r to m inim ize the loss of volatile com pounds, the
sam ple s we re not analyze d will be save d at 4 o C.
- Inte rnal standard calibration and standardization
Inte rnal standard calibration is a fam iliar approach to
q u a n tita tive a n a lys is o f s ta tic h e a d s p a c e ga s
chrom atography; this m e thod allows ove rcom ing for any
deviation caused by m atrix effects and injection conditions
of gas chrom atography. Before the separation by extraction,
a known concentration of analyte is added to each standard
solutions and sam ple. That com pound is called the internal
standard. The choice of an internal standard that is suitable
for the analysis of diffe re nt analyze s is conside re d the
im portant part of the inte rnal standard calibration. Inte rnal
standard com pound m ust be pure form and doe sn’t e xist
in the sam ple s, at le ast at in the analyze d are a, according
th e s e lim ita tio n s it w a s c h o s e n , a n a p p ro p ria te
conce ntration inte rnal standard, using re age nt n-butanol.
The sam e pe rce ntage of the inte rnal standard solution for
e ach standard and sam ple analyze d should be adde d by
using this m e thod. For accom plish of calibration curve ; 3
m L of (4 g/L) internal standard be added to 2 m L of standard
solution into a 10 m L vial .Pre pare 4 points of e thanol
2970 http://www.revistadechimie.ro
analytical standards, using 200 proof e thanol, ranging from
10 to 200 g/L, vial is se ale d, sam ple s will place in the ove n
at te m pe rature 70o C for 15 m in, m ore volatile com pound
(e thanol), which re sults from he ating the sam ple will rise
to the he ad are a until the e quilibrium state is re ache d,
1m L of the he a dspa ce volum e w a s ta ke n for inje cte d
dire ctly into a GC w ith tightly syringe , 1-2 inte rm e diate
calibration we re pre pare d as ve rification solutions from
the se parate e thanol source , the n can be account e thanol
conte nt by form ing calibration curve by plotting the ratio of
the analyte signal (are a of the pe ak) to the standard signal
as function to analyte concentration of the standards, figure
2 show that.
Instrum e ntation and GC condition
In this study GC (Shim adzu, Japan, 2014) w as use d,
which was e quippe d with com pute r inte grator software
(GC solution, Shim adzu) and the flam e ionization de te ctor
(FID). Hydroge n and air flow rate we re se t at 30 and 300
m L/m in, re spe ctive ly. Te m pe rature of the FID de te ctor and
the inje ction port was se t at 250, and 120o C, re spe ctive ly.
The flow rate of nitroge n is 6 m L/m in, split ratio is 2, and
c o lu m n flo w is 1m L u s e d a s th e c a rrie r ga s . ZB-1
(phe nom e nx) se paration colum n (L: 30 m , ID: 0.32 m m ,
HP-5, 5% phe nyl m e thyl siloxane capillary colum n from
Phenom enix with a low-film thickness of 0.5µm ) was use d
to p e rfo rm in th is stu d y. In th e b e gin n in g: th e o ve n
tem perature was set at 40oC for 4.5 m in, and then increased
to the final te m pe rature of 120o C for 1 m in at the rate 40o C/
m in. The volum e of injection was 1m L. Split injection m ode
w a s s e le c te d . Th e 10 m L h e a d s p a c e via ls a n d th e
alum inum crim p caps we re obtaine d from Phe nom e nix.
The 1000 µm L m icro pipe tte we re obtaine d from Dragon
(Shanghai, China).
Statistical analysis
All the data use d for m e thod validation we re analyze d
according to SPSS 14.0, and the data use d for qualitative
analysis. The statistical significance le ve l was se t at P <
0.05.
Re s ults and dis cus s io ns
This type of colum n (ZB-1, 40 m e te r) w a s se le cte d
be cause we inte nd to know the ability and the range of
e tha nol conce ntra tion w hich ca n d e te rm ine by using
standard solution of ethanol and real solution that represent
Fig. 2. Re te ntion tim e of e thanol (2.73 m in) :
n- butanol use d as inte rnal standard solution
REV.CHIM.(Bucharest)♦ 69 ♦ No. 11 ♦2018
of bioe tha nol in fe rm e nte d broth w he n w e use sta tic
h e a d spa c e ga s c h rom a togra ph y ((SH-GC)) te c h n iq u e
inste ad of dire ct inje ction of liquid.ZB-1 (100, 150 m e te r)
colum n is the m ost popular colum n for the de te rm ination
of e thanol conte nt of de nature d fue l e thanol by dire ct
inje ction of liquid [16, 17]
The GC conditions use d which is de scribe d in m ate rials
and m e thods are se le cte d afte r m any tim e s of runs for
re aching the be st re solution, the re te ntion tim e of e thanol
w a s 2.73 m in a n d n - b u ta n ol w h ic h u se d a s in te rn a l
standard solution was 7 m in as shown in figure 2.
Validation of the analysis m e thod
A pure e tha nol solution (99.9% w /w ) w a s m ix w ith
distille d wate r to ge t e thanol conce ntrations (200, 100, 50,
25, 12, and 10 m g/m L). Each dilute d solution sam ple (2
m L) w as poure d into a 10-m L cape d vial. Afte r m ixing
w ith (3 m L) of a (4m g/m L) inte rna l sta nda rd spiking
so lu tio n (e q u iva le n t to 12 m g), via l is tigh tly c lo se d ,
sam ple s will place in the ove n at te m pe rature 70 o C for
tim e 15 m in, volatile com pound (e thanol) in the sam ple
will partition into the he adspace until re ache s e quilibrium
state , 1 m L of the he ad space phase was dire ctly inje cte d
into a GC with syringe. All concentrations of were m easured
in thre e tim e (triplicate ).
Re lative standard de viation (RSD) or calle d coe fficie nt
of variation (CV) w as use d to e valuate pre cision w hich
obtaine d of e ach inte rnal standard solution have the sam e
conce ntration at a rate of thre e tim e s of e ach sam ple ,
which was le ss than 5% for m ost sam ple s.
Re lative e rror of the m e an (REM) was use d to m e asure
th e a c c u ra c y, Th is re la tio n s h ip w a s c a lc u la te d a s
diffe re nce be tw e e n m e asure d value and true value of
c o n c e n tra tio n s d ivid e d b y tru e va lu e b y a ga s
chrom atograph, ge ne rally (REM) was le ss than 4% for all
sam ple s.
Re la tive re s p o n s e fa c to r a n d q u a n tita tive e th a n o l
de te rm ination
Take n 2 m L of e thanol at (200, 100, 50, 25, 12.5 and 10
m g/m L) and 3 m L of (4 m g/m L) inte rnal standard solution
we re poure d into an 10 m L vial, afte r that the sam ple is
putte d inside a cle an vial and the vial is tightly se ale d,
sam ple s will place in the ove n at te m pe rature 70 o C for
tim e 15 m in, volatile com pound (e thanol) from the sam ple
will partition into the he adspace until a state of e quilibrium
is reached, 1m L of the head space of sam ples was injected
dire ctly into a GC with tightly syringe . It will be ge ne rate d a
re gre ssion line from the GC pe ak are a unde r curve (A)
ratio of e thanol to n-butanol (y-axis) (AS/AIS), against the
conce ntration ratio of e thanol (x-axis) (CS), and (fig. 3)
show that.
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Table 1 shows the re lationship be twe e n e thanol conte nt
in the standard solution with (AS/AIS), (RSD) %, (REM) %.
The slope of the re gre ssion line w ill re pre se nt re lative
re sponse factor (RRF) is the , as in the e quation (1):
RRF = (AS/ AIS) ÷ CS (1)
And the n e tha nol conte nt w a s ca lcula te d using the
following e quation:
CS(m g/m L)= (AS/AIS)x1/(RRF) (2)
whe re AS is the e thanol pe ak are a unde r curve ; AIS is n-
buta nol pe a k a re a unde r curve ; a nd CS is the e tha nol
conte nt (m g/m L).
Fig. 3. Line ar Re gre ssion of Calibration curve of e thanol
conce ntration w ith (AS/AIS)
GC Conditions
For se le ction of the GC conditions, it has be e n se le cte d
n-butanol as inte rnal standard be cause of the se re asons;
first, n-buta nol a s che m ica l structura l is sim ila r to the
analyte (ethanol), and the it will undergo to sam e extraction
c o n d itio n s a n d c h ro m a to gra p h y, o th e r w is e th e
com pe nsation will be lost, se cond n-butanol available in
extrem ely pure form and never appear in the sam ples third
it has re te ntion tim e (3.5 m in) diffe re nce about e thanol
re te ntion tim e (7.3 m in) in the chrom atogram . As shown
in figure 2.
For choosing of e thanol conce ntration it was the highe st
conce rtino 200 m g/m L and the lowe st conce ntration was
10 m g/m L. This se le ction de pe nd on fact that the pe rce nt
of e thanol in fe rm e nte d broth by ye ast of saccharom yce s
le ast than 20% and the conce ntration of e thanol le ss than
1% ha s d e form c hrom a togra m . Also for c hoosing the
conce ntration of inte rnal standard solution we should to
m atch be twe e n conce ntration and the volum e of inte rnal
standard was adde d to vial with standard and re al solution
to ge t the be st re solution without change the volum e of
internal standard solution for ethanol content range depend
on this conce pt the be st conce ntration of inte rnal standard
w as 4 g/m L. The re st of othe r conditions, w e re initially
de pe nde d on pre vious studie s [17, 18], until we re re ache d
the be st conditions. As m e ntione d be fore .
Table 1
ETHANOL CONTENT USING GAS
CHROMATOGRAPHY BY STATIC
HEADSPACE GAS CHROMATOGRAPHY
METHOD WITH (RSD) AND (REM)
Table 2
ETHANOL CONTENT DETECTION IN
FERMENTED BROTH
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Determ ination of ethanol concentration in ferm ented broth
Five sam ples of ferm ented broth were taken at different
tim e (6, 12, 24, 48, 72 h) for de te rm ina tion of e tha nol
conte nt by he ad space gas chrom atography as m e ntione d
before in sam ple preparation and the results shows in table
2 as follow.
Co nclus io ns
Utilizing a he adspace sam pling-gas chrom atography
w h ic h is m ixe d w ith a n in te rn a l s ta n d a rd fo r th e
de te rm ination and quantification of e thanol conte nt in
fe rm e nte d broth m e dium sam ple s in the laboratory se e m s
to be very prom ising in the analysis of volatile com ponents.
Colle cte d data in this re se arch will he lp to m onitor the
fe rm e ntation proce ss and de te rm ine the conce ntration of
e thanol in biofue l. Although ZB-1 colum n (40 m e te r) is
sm a lle r tha n com m on (ZB-1) colum ns w hich use d to
determ ine the ethanol in the solutions but this colum n show
that it has a good pe rform ance unde r ce rtain condition for
de te ction of e thanol. It can be use ful for the analysis of
e thanol in biofue l sam ple s.
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Manuscript re ce ive d: 27.02.2018
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