J Pack. Sci. TechnoL Vol. 2 No. 1 (1993)

A Fast Gas Chromatographic Method for the Separationof Nitrogen, Oxygen, Carbon dioxide and Argon and
Its Application to In-package Modified AtmosphereTakashi HIRATA*. Takeo NISHIYAMA**, Hiromi SATO*,
Yutaka ISHIKAWA*, Takeo SHIINA*** and Takasuke ISHITANI****

A gas chromatographic method was developed for the rapid separation of headspacegases in film-package of fresh produce. A balanced set of three columns simultaneously
separated four gases including N 2 , O 2 , CO 2 and Ar. The gas composition was accurately
c a l c u l a t e d b y me a s u r i n g t h e r a t i o o f e a c h p e a k a r e a o n t h e c h r o ma t o g r a m s a n dconsidering the relative response of a thermal conductivity detector to each gas. Thismethod was found to be useful for the accurate determination of O 2 and CO 2 in themodified atmosphere, because Ar proved to be kept in film packages of the produce ata concentration above ca. 0.9% in various cases while Ar overlaps O 2 on the gas
chromatograms in conventional gas chromatographic systems.
Keywords : Argon, Oxygen, Carbon dioxide, Fresh produce, Headspace gas, MA packaging, Gas chromatography, Gas composition

I NTR ODUC TI ON

distribution. Since a variety of technicalinformation including permeability of films,

Modified atmosphere packaging (MAP) has
been studied as a technique to prolong theshelf-life of fresh vegetables and fruits1)2} . The
technique basically expects self-equilibrationof the in-package gas composition, which may
provide a suitable atmosphere for controlling
excess respiration of the fresh produce duringresp ira tion rate s in a given O 2 / CO 2 at mo sphere, respiration quotients, free volume ofthe package, etc. are required for the proper
design of MAP, computer aided simulation is
one of the most promising techniques toobtain suitable MAP conditions 3)～8) .
F o r a n y M A P d e s i g n , t h e e x a ct d e t e r mi -*National Food Research Institute, 1 - 2, kannondai - 2, Tsukuba, 305 Japan **Okayama Branch of Kobe
Center for Quality Control and Consumer Service, Ministry of Agriculture, Forestry and Fisheries, 24,
Tenjincho - 9, Okayama, 700 Japan ***Hokkaido National Agricultural Experimental Station, Kyusen - 4,
Shinsei, Memuro, Kasai - gun, Hokkaido, 082 Japan ****National Agriculture Research Center, 1 - 1, Kannondai - 3, Tsukuba, 305 Japan- 1 5 -

Gas chromatographic evaluation of MA within film packages

nation of the in-package concentrations of N2,
O2, CO2 and Ar is necessary, because either
anaerobic conditions or the presence of excess

O2 and overlaps O2 on gas chromatograms.
However the presence of Ar in MAP should

CO2 results in the development of off-odors,

because in many cases the critical levels of
0a for providing proper MA conditions areless than 5%.
The objectives of this paper are to develop
a gas chromatographic method for simultaneously measuring concentrations (%) of O2,
N2t CO2 and Ar in MAP, and to evaluate thegas exchange in MAP of fresh produce on

and also the higher concentration of O2 allows
for a rapid senescence. Because of the demands for the rapid and simultaneous determination of the in-package gas composition in
MAP, gas chromatographical methods havebeen used. Brenner9) described a combinationof silica gel and molecular sieve 5A as parallelcolumns. Karel et al10). used columns packed
with silica gel and a molecular sieve that wereconnected in series. Recently, Yamashita etal11) used a combination of activated charcoal
and a molecular sieve as a parallel column

be considered for establishing the MAP system

the market or during storage using themethod developed.
MATERIALS AND METHODS
1. Vegetables and packaging

system and calculated the gas composition
from the ratio of the peak areas. Aerograph
A90 - P36) or Alltech CTR I3)12) have been also

Locally grown broccoli was purchased at
a wholesale market, packaged in plastic films
and stored at 15°C and 90%rh. The character-used for analyzing the gas composition, while
even in some recent papers 13)15) , the gas

istics of the films used and the packaging

analysis procedures are not given in detail.
In these columns, Ar is not separated from- 1 6 -

conditions are listed in Tables 1.
Fresh vegetables packaged using flexible

/ . P a c k . S c i . Te c h n o l Vo l . 2 N o . 1 ( 1 9 9 3 )plastic materials were purchased at a local
supermarket. The packaging materials wereas follows : A stretch film of polyvinyl chloride
for shiitake fungus (Lentinus edodes Sing.),
shimeji fungus (Pluribus ostreatus Quel.) andcucumber, low density polyethylene for mungbean sprout (Vigna radiata Wilcz.), a mix ofshredded vegetables including mung bean
sprout, cabbage, carrot and black fungus

Fig. 1 A schematic diagram of the column systemsimultaneously separating four gases.

{Auricularia auricula-judae Quel.), and polypropylene for carrot,
2. Gas chromatography
A gas chromatograph (GC - 7A, Shimadzu,

kuropack 54 was separately cooled down to-80°C by soaking the column in dry icemethanol. Temperatures of the TCD and injection port were adjusted to 90°C. The flow

Japan) equipped with a thermal conductivity
detector (TCD) was used to analyze N2 , O2 ,CO2 and Ar in a gas mixture. Carbon dioxide
was separated from the mixture by a stainlesssteel column (3mm × 0.5m) packed withactivated charcoal (mesh : 60～80). Oxygen, N2

rate of the carrier gas (He) was 60ml/min.
Peak areas in a gas chromatogram were
measured using a Shimadzu C-RIA integrator.
The packing materials for columns were allpurchased from GL Science (Tokyo).

and Ar were separated by a packed column

3. Gas analysis(3mm × 6m) of Gaskuropack 54, (a styrene

About 0.3ml of the headspace gas wasperiodically sampled from the package andwas injected in the gas chromatograph. A/divinyl benzene copolymer, mesh : 60~80),
while CO 2 was absorbed in the packing
material. Both columns were linked in parallel

small silicone rubber piece (ca.l0mm × 10mm)
was glued on the surface of the package sothat the gas sampling was easily carried out

so that simultaneous analysis of the fourgases were carried out using a single injectionof the gas sample. For controlling the split
ratio of the gas flow at a diverging pointof the linked column, a packed column (3mmby direct sticking of a syringe needle into
the package.
4. Calculation of gas composition
The gas chromatographic profile of a gas
mixture including N2, O2, CO2 and Ar is sche-×4m) of Uniport B (mesh 60~80) was addedin series to the activated charcoal column.
The total column system is shown in Fig. 1.

matically given in Fig. 2. Peak A to B and
Peak C to F indicate the gas portions separated by the columns packed with activatedcharcoal and Gaskuropack 54, respectively .

Three meter column of Uniport B was also
connected to TCD as a reference column.
While Uniport B and activated charcoal were
controlled at 70°C in the column oven, Gas-

- 1 7 -

J. Pack. Sci. Techno!. Vol. 2 No. 1 (1993)

tion. But the analysis using the simply linked
column system proved to be impracticalbecause the gas groups (O2 + N2 + Ar/CO2, N2

RESULTS AND DISCUSSION
1.Gas chromatograms
A clear separation of CO2 from a gas mixture including N 2 , O 2 , CO 2 and Ar were/O2/Ar) showed significantly different
retention times. It is natural to consider that

performed by a single column (3mm × 0.5m)

Gaskuropack 54 (3mm×6m) at~80°C (Fig.

a difference in the column lengths may cause
a pressure imbalance between the insides of
the two columns resulting in the different
retentio n times.

3). These single columns made it possible toseparate the gases within a short retention
time. However, for determining four gases,
two measurements were required and thus

Therefore, Uniport B was connected to maintain a balance between the retention times.
The gas mixture was analyzed on the system
and a typical gas chromatograph of the gas

careful calibration of an absolute amount of

mixture is shown in Fig. 4b. Five different

the injected gases was very important forhaving accurate results. Therefore, in order
to simultaneously analyze the four gases, thetwo columns were linked in multiple connec-

peaks were successfully observed. Each peak
was assigned from its retention time as A (apacked with activated charcoal, while O2 , N2
and Ar were separated from each other on

Fig.3

mixture of N2, O2 and Ar), B (CO2), C (N2),
D (O 2 ) or E (Ar). The optimum length ofFig.4

A gas chromatographical profile
of a laboratory atmosphere on
a Gaskuropack 54 column.
The atmosphere was analyzed
on a single Gaskuropack column(3mm×6m) at -80°C.

-19-

Effect of column length oi uniport B on
the resolution and retention time ofinorganic gas peaks.
Column lengths of Uniport B: (a) 1m,(b) 4m, and (c) 5m. Symbols are thesame as those in Fig.2

Gas chromatographic evaluation of MA within film packages

where C, D, E and F are the areas of Peaks
C, D, E and F, respectively.
B e ca u se b o th P ea k B / A a n d p e a k F / ( C+D+E) express the ratio of CO2 to a mixtureof N 2 , O 2 and Ar, the area under Peak F iscalculated from equation (5),BF =A
RETENTI ON

Fig.2

TIME

-

A schematic chromatogram expressing thecomplete profile of the inorganic gases

where A and B are areas of Peaks A andB, respectively. By substituting equation (5)into equations (1) to (4), the gas compositions

including O2. N2, CO2 and Ar.
A:O2+N2+Ar.
E:Ar,

B:CO2,

C:N2,( C + D + E ) ・・・・・・・・・(5)

D:CO2,

F:CO2

given by the volume percentage (%) wereobtained. It is not necessary to consider that
Peak A and B correspond to a mixture ofthree gases (N2, O2 and Ar) and CO2 respectively, because activated charcoal can not absorb
N 2 , O 2 and Ar at all. Each Peak from C to
F corresponds to N2, O2, Ar and CO2, respectively. Peak F is provided as a dummy forconvenience of calculation because Gaskuro-

the split ratio of the gas flow at the diverging point varies because the gas compositionis always calculated from the ratio of eachpeak area (C~F) to the total peak area (C+D+E+F).
5. Relative response of TCD
The relative response of TCD to an inor-

pack 54 strongly absorbs CO 2 and thus noganic gas was independently calibrated bydetermining the peak area as follows :An accurate amount (0.5ml) of authentic

peak can be obtained from CO 2 .
If it is assumed that the thermal detector
shows the same response to every gas, thegases (O2 99.99% ;N2, CO2, Ar 99.999%) was

gas composition (%) can be expressed asfollows ;injected in the column system and total peakareas were calculated by the integrator. The
averaged value of five measurements was
obtained. After (C +D+E)/A (D +E～0) was
calculated from the analysis of the authentic
N 2 , the peak area of CO 2 corresponding toPeak F was calculated by equation (5), andthen B+F was considered as the total areaof CO2.

- 1 8 -

Gas chromatographic evaluation of MA within film packages

Uniport B for separating the gases on theplastic films commonly used for this system
are flexible. Therefore, the gas composition
inside the package is easily obtained bymeasuring the ratio of each peak area (C, D,
E and F) to the total peak area (C+D+E

column system was 4m (Fig. 4b). When thecolumn was shorter than 4m, the retention
time for N2, O2 and Ar increased significantly(Fig. 4a). On the other hand, if the column
was longer than 4m, CO2 and the gas mixture+F). Table 2 shows the gas composition oftended to overlap N 2 , O 2 and Ar (Fig. 4c).

the outside atmospheric air calculated from

The advantage of this method is that it
may allow one to inject an approximate
amount of a gas mixture for determining thegas composition, and a calibration curve using
authentic gases is not required.

the ratio of peak area o f the gas ch ro matogram. The calculation procedure alreadymentioned did not give a reasonable result
for the composition when compared withwidely recognized values 16) . Therefore, therelative responses of TCD to the gases on
helium were determined. Table 3 shows theclear differences for the relative responses
with small standard deviations among the fourgases. A similar result has been reported by
Messner et al, 17) covering a range of 30°C

2. Gas composition
Several authors15) calculated the gas composition from areas or heights of the peaks
using calibration curves of the standard gases.
This procedure requires careful injection ofan accurate amount of gas sample, and none

to 160°C of TCD. When the observed peakareas were divided by the relative response
values, and then the gas composition of theof them mentions the presence of Ar in thea i r . W h i l e t h e p a r t i al p r e s s u r e o f e a c hinorganic gas in a MAP system fluctuates

air was calculated using equations (1) to (5),the results proved to be highly acceptable(Table 2), indicating that the discrepancy

drastically, especially during the early stageof storage, the integrated gaseous pressure
inside the package is stable and is equal tothe air pressure outside the package because

between the accepted values and the experi
mental values obtained this time was mainly

Table 2 The atmospheric gas composition (%) analyzed by gas chromatography.
Values widely
Correction by TCD sensitivity to each gas
Gas
accepted*corrected

not corrected

Nitrogen

78.08

78.95

OxygenCarbon dioxide

20.90

1 9.93

20.99

0.05
0.98

0.06
1.07

0.03
0.94

Argon

* H u m p h r e y , 1 9 2 6 1 6 )- 2 0 -

78.03

J. Pack. Sci. TechnoL Vol. 2 No. 1 (1993)

O 2 or Ar concentration during storage.
When compared with other conditions, theO2 oncentration under Condition A was kept
at a higher level even after 7 days. Under
Condition B, O2 gradually decreased to become
equilibrated at ca. 2% after 48 hour storage

Table 3 Relative responses of TCD to inorganic
gases on helium.
Relative response'
Gas
AverageStandard deviation

Ni trogen
Oxygen

100
94.3

0.57

Carbon dioxide

115.9

1.85

at 15°C. In both conditions, color, which is

Argon

107.0

0.56

important as the quality determinants of thevegetable, decreased as a function of storage
time. Under Condition C, the O2 concentration
reached after 48 hour storage, and thereafter
the respiration proceeded anaerobically, result-*Average of five measurements

due to the gas-dependent response of theTCD.

ing in the generation of undesirable off-odors.
But in any condition, Ar was kept in therange of 0.9 to 1.0%. If Ar had overlapped
O2 on the chromatograms, the observed con-

3. Gas composition within MAP
Changes in the gas composition in MAP
of broccoli are given in Fig, 5. The packaging
conditions A and C in Table 1 apply to
extreme cases, because these conditions were
determined to intensify the changes in either

centrations of O2 , even under Condition C,
would have showed almost 1 % O 2 duringstorage.
Some researchers have suggested that 2~
5% O2 and 6～10% CO2 are recommended forthe storage of broccoli under CA/MA 18)19) .
Undesirable off- odors were formed frombroccoli when the produce was stored under
the CA with very low O2 (0.25% or less) at
5°C 20) . The off-odors were also developed
under 1% or less O2 in MAP21). However, insome cases, the off-odors were also developed
under relatively high concentrations of O2 (<0.5-4 %) 21)22} .

0

1

2

3

4

5

STORAGE TIME, DAYS

Fig. 5 Changes in O 2 , CO 2 and Ar in film packages
during the storage of broccoli at 15°C.
The packaging conditions are given in Table1.

Condition A (PMP)
Condition B (LDPE)
Condition C COPP)It seems that these discrepancies in dividing
the concentration between aerobic and anaerobic respirations are due in part to no
separation of Ar from O 2 .
Table 4 shows the gas composition in MAPof several vegetables purchased from a local
supermarket. The O 2 concentrations were in- 2 1 -

Gas chromatographic evaluation of MA within film packages

Table 4 Modified atmospheres observed in film packages for fresh produce purchasedfrom a local supermarket.

* Folyvinyl chloride** Low density polyethylene""A mix of mung bean sprouts, cabbage, carrot and black fungus.

the range of 5～16%, while the Ar concentrations were in the range of 0.9 to 1.0%
again. The proper concentrations of O 2 andCO 2 in MAP were 1～2% and 10～14% fors h i i t a k e f un g u s at 1 ℃ 2 3 ) a n d 3 - 6 % f o r
tomatoes at 10°C24), respectively. The O2 concentrations for keeping the quality of cucumbers were 3-5% at 8°C 19) . Many other vege
tables showed similar values of O2 levels forproper packaging. Generally, the rate ofrespiration sensitively changes below about
8% of O2 and above about 1% of CO2, whilea low O 2 enriched with CO 2 beyond theconcentration of tolerance, induced anaerobic
respiration and/or CO 2 injury 2) .
Therefore, the respiration of the vegetables
shown in Table 4 was very lightly suppressed but anaerobic respiration was not yet
proceeded at all. It can be said that if theO 2 concentration had been lower, a longer
shelf-life for the vegetables would have beenobtained. However, if the packaging designof MAP is not based on an accurate evalua-- 2 2 -

tion of O 2 concentration, a slight change inthe storage condition may cause anaerobic
respiration because suitable O2 concentrations
for the vegetables are 1~6% as previously
mentioned.
Determination of O2 concentration in MAP
has used the packed column with a molecularsieve9)～11)22). However, the residual concentration of O 2 may have been overestimatedbecause Ar always overlaps O 2 on the gas
chromatogram in their systems. Therefore, theindividual analysis of O 2 and Ar using thesystem presented herein are important. Alternatively, because it is noticed that in manycases Ar concentration is kept in the levelof 0.9～1.0% as shown in Table 4 and Fig.
4, one percent O2 should be subtracted fromthe values obtained by the conventional
column systems.
In conclusion, the gas chromatographic pro
cedure developed in this paper can be applied
to separate the atmospheric inorganic gases
from each other by a single injection and/ . P a c k . S cl . Te c h n ol Vo l . 2 N o . 1 ( 1 9 9 3 )determine them accurately. It can be expectedthat this technique is applicable to designfor other packaging systems such as gas
replacement packaging and O 2 absorberpackaging as a tool for the optimization ofthese packaging systems.

ASAE.T 30 (2), 548 (1987)
13) F. Carlin, C. Nguyen-the, G. Hilbert and Y.
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15) A. Ballantyne, R. Stark and J. D. Selman, Int.
J. Food Sci. and Technol., 23, 267 (1988)
16) W. J. Humphreys, 1926. "International Critical

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