Chemical Method for Determination of Carbon Dioxide Content

in Egg Yolk and Egg Albumen

1,2
K. M. Keener,*
,3
J. D. LaCrosse, and J. K. Babson
*Department of Food Science, North Carolina State University, Raleigh, North Carolina 27695-7624; Department of
Chemical and Biochemical Engineering, University of Iowa, Iowa City, Iowa 52242; and Department of Biological and
Agricultural Engineering, North Carolina State University, Raleigh, North Carolina 27695-7625
ABSTRACT The safety, quality, and shelf life of shell
eggs is a functionof carbondioxide content. Acommercial
process was recently developed for rapidly cooling shell
eggs by using cryogenic CO
2
. The benet of this new
process over existing cooling processes is that the CO
2
addition during cryogenic cooling provides additional
safety and quality enhancements. In order for these bene-
ts to be fully developed into a process that can be
adopted by the egg industry, and thus realized by the
consumer, the amount of CO
2
absorbed by the egg during
this process needs to be quantied. Because the albumen
pH of rapidly cooled eggs was reduced to pH <6.5, ex-
isting titrametric methods were not adequate for de-
termining CO
2
content. They did not prevent CO
2
loss
during neutralization. A simple and accurate method for
determining CO
2
content in acidied egg albumen and
yolk samples was developed. This method involves the
(Key words: carbon dioxide, egg yolk, egg albumen)
2001 Poultry Science 80:983987
INTRODUCTION
The safety, quality, and shelf life of shell eggs is a
function of carbon dioxide content. Acommercial process
was recently developed for rapidly cooling shell eggs
using cryogenic CO
2
. The benet of this newprocess over
existing cooling processes is that CO
2
addition during
cryogenic cooling provides additional safety and quality
enhancements. In order for these benets to be fully de-
veloped into a process that can be adopted by the egg
2001 Poultry Science Association, Inc.
Received for publication August 28, 2000.
Accepted for publication February 25, 2001.
1
Journal Paper No. FSR 00-12 of the North Carolina State University
Department of Food Science Journal Series.
2
This paper was presented at the Institute of Food Technologists
Annual Meeting Paper No. 51G-6 at the IFT Annual Meeting in Dallas,
Texas, fromJune 10 to 14, 2000. The use of trade names in this publication
does not imply endorsement by the North Carolina Agricultural Re-
search Service of the products named or criticism of similar ones not
mentioned.
3
To whom correspondence should be addressed: kevin_
keener@ncsu.edu.
983
liberation of CO
2
from an acidied egg sample into a
standardized, dilute sodium hydroxide solution inside a
sealed jar. The egg sample and a small beaker containing
the standardizedsodiumhydroxide solution are placedin
a glass jar andsealed. Next, a concentratedacidphosphate
solution is injected through a rubber septum in the cap
of the jar onto the egg sample, while avoiding contact
with the sodium hydroxide solution. The sample is then
stored at 37 C for 24 h. During this storage period, the
carbon dioxide is released from the egg sample and is
absorbed into the sodium hydroxide solution. After-
wards, the dilute sodium hydroxide solution is removed
and titrated to the phenolphthalein endpoint using a di-
lute, standardized hydrochloric acid solution. The
amount of hydrochloric acid solution required for neu-
tralization can be directly related to CO
2
content in the
sample.
industry, and thus realized by the consumer, the amount
of CO
2
absorbed by the egg during this process needs to
be quantied.
There are reference methods available to measure CO
2
content in a number of foods and food products, such as
beer (Ofcial Methods of Analysis, 1995a), baking powder
(Ofcial Methods of Analysis, 1995b), and water (Ofcial
Methods of Analysis, 1995c), but there is currently no
published method for CO
2
measurement in egg albumen
or egg yolk. The recent application of cryogenic CO
2
gas
to shell eggs for rapid cooling has necessitated the devel-
opment of an economical method to monitor CO
2
levels
in shell egg components. Because of the viscous nature
of egg components, direct measurement of CO
2
using an
ion selective probe does not accurately measure dissolved
CO
2
content. Calibrated near- infrared systems and gas
chromatography systems are adaptable for CO
2
analysis;
however, these systems are expensive and require peri-
Abbreviation Key: PT phenolphthalein; KPH Potassium phthal-
ate hydroxide; mEq milliequivalents.
KEENER ET AL. 984
odic calibration. Historically, titration procedures have
been used for measuring CO
2
and carbonate species in
eggs and egg components (Mueller, 1958; Reinke and
Baker, 1966; Heath, 1977), and the proposed method for
CO
2
analyses of acidied egg albumen and egg yolk also
requires a titration step. The main improvement of this
method is the ability to prevent CO
2
volatilization during
titration. Cotterill et al. (1959) showed that directly titrat-
ing an acidied egg albumen sample to a neutral pH
resulted in a CO
2
volatilization. With increased use of
carbon dioxide in foodproducts, it is necessary to develop
an inexpensive method of CO
2
analysis that can be used
on acidied samples of less than 10 g.
The procedure developed for determining the CO
2
con-
tent in acidied egg albumen and egg yolk was adapted
from a method developed by Fleming et al. (1974) to
measure CO
2
levels in cucumber brines. The process in-
volves driving CO
2
out of the albumen and into a stan-
dardized NaOH solution by using a high ionic strength
acid phosphate solution (pH <1). The NaOH solution is
then combined with BaCl
2
and nally titrated with HCl
to determine CO
2
concentration. The objective of this
study was to evaluate the proposed method for accuracy
and precision in measuring CO
2
content in egg albumen
and egg yolk.
MATERIALS AND METHODS
For determining method accuracy, a 2 3 6 random-
ized block design experiment was setup for determining
CO
2
concentration in egg albumen and egg yolk. Eighteen
white eggs, approximately 30 d old, were purchased from
a local grocery store. The eggs were broken out, and
yolks and albumens were pooled. Duplicate samples of
albumen and yolk samples consisting of approximately
0, 2, 4, 6, 8, and 10 g with additions of 0, 2, and 4 mL of
a 1.1 mg/mL CO
2
solution were made and evaluated
using this chemical method. The CO
2
solution was added
to cover the range of CO
2
expectedin cryogenically cooled
eggs. The maximum solubilities of CO
2
in water at 0 and
20 C under one atmosphere (101.3 kPa) of CO
2
are 0.33
and 0.17%, respectively (Brey, 1978).
For determining experiment precision, six 2-d-old eggs
from Hy-line chickens were collected and broken out.
Yolks and albumens were pooled. Five 6-g samples
were tested.
Samples were prepared by placing a 30-mL glass jar
4
containing 12 mL of standardized, dilute NaOH solution
(0.15 N) into a 250-mL glass jar.
4
A measured amount
of albumen was then placed into the 250-mL glass jar,
and a screw-cap lid was placed on the jar. The screw-cap
lid contained a 1.25-cm rubber septum
4
and a plastisol
liner. Figure 1 shows details of the apparatus. The lid
was heated in a drying oven at 50 C for approximately
3 min, before placing on the jar, to soften the liner and
4
Inmark Inc., Atlanta, GA 30336-0309.
FIGURE 1. Equipment used to determine CO
2
concentration in albu-
men and yolk samples.
provide a good seal. The CO
2
solution, if added, was then
injected through the septum onto the top of the albumen
or yolk sample. Next, 15 mL of the acid phosphate
(NaH
2
PO
4
H
2
O + H
3
PO
4
) solution (see below) was in-
jected onto the albumen. The acid was not allowed to
splash into or otherwise contact the standardized NaOH
solution. When the acid phosphate solution was added
to the albumen, the carbonate (CO
3
2
) in the albumen
was converted to CO
2
. The high ionic strength (pH <2.5)
reduced the solubility of CO
2
, which was driven into the
12 mL of standardized NaOH in the 30-mL vial. The
NaOH reacted with CO
2
as shown in reaction 1:
CO
2
+ 2 NaOH Na
2
CO
3
+ H
2
O. [1]
From this reaction, it can be shown that 22 mg of CO
2
reacts with each millequivalent (mEq) of NaOH. Back-
ground CO
2
measurements were performed using exactly
the same procedure except no sample was added. After
24 h at 37 C, the vials were removed from the jars. For
this investigation, storage at 37 C for 24 h was found to
provide 100% CO
2
recovery for the samples tested and
was selected for convenience. The earlier study on cucum-
ber brines (Fleming et al., 1974) indicated that various
incubation times and temperature combinations could be
used to produce 100% recovery of CO
2
in cucumber
brines, but these were not investigated in this study. After
24 h, the jars were removed from the incubator, and the
30-mL vial containing the standardized NaOH solution
was removed from each jar. The vials were then random-
ized and analyzed. For this analysis, 3 mL of 1 N BaCl
2
and one drop of phenolphthalein (PT) indicator were
added to each vial and reaction 2 occurred:
Na
2
CO
3
+ BaCl
2
2 NaCl + BaCO
3
. [2]
The excess BaCl
2
reacted with the Na
2
CO
3
to form NaCl
and BaCO
3
precipitate. This step removed all of the
Na
2
CO
3
. This solution was then titrated to the PT end
point with a standardized HCl solution (0.15 N). The
mEq of NaOHremaining was calculated fromthe amount
of HCl used in the titration. Equation 3 was used to deter-
mine the quantity of CO
2
in the albumen.
CHEMICAL METHOD FOR DETERMINATION OF CARBON DIOXIDE IN EGGS 985
mg of CO
2
g albumen

[mEq of NaOH in solution mEq HCl used] [3]

mg of CO
2
mEq
1
1
]

1
g of alabumen
1
1
]
_

,
.
Reagents
Acid Phosphate Solution. Dissolve 200 g of NaH
2
PO
4
H
2
O and 30 mL of 85% H
3
PO
4
in 600 mL of deionized
water. Slowly add 63 mL concentrated H
2
SO
4
and dilute
to 1 L.
Indicator. Dissolve 1 g PT in 100 mL ethanol.
NaOH Solution (0.2 N). Dilute 8 g of NaOHto 1 L with
deionized H
2
O. Weigh a nominal amount of potassium
phthalate hydroxide (KPH FW 204.228), approximately
0.75 g, and dilute to about 10 mL with deionized H
2
O.
Add one drop of PT indicator to the KPH solution and
titrate with NaOHsolution. Equation 4 was used to deter-
mine the normality of the NaOH.
Xg
204.228 g/gEq
[4]
1000 mEq/gEq
YmEq
ZmL
Normality
where X is the actual grams of KPH used, Y is the mEq
of KPH, and Z is the milliliters of NaOH used to reach
PT endpoint.
HCl Solution (0.15 N). Dilute 1 N HCl to produce a
0.15 N solution. Add one drop PT indicator to the HCl
solution, and titrate with standardized NaOH solution to
determine normality (Equation 5).
XmEq
mL
YmL
ZmEq
WmL
Normality [5]
where X is the normality of NaOH solution, Y is the
amount of NaOHsolution required to reach PT endpoint,
Z is the resulting mEq of the NaOH solution, and W is
the amount of HCl used in the titration. This procedure
was performed three times on each HCl solution, and
the average normality was determined. This average was
used in further calculations.
CO
2
Solution (1 mg CO
2
/mL Solution). Dilute 0.1909
g NaHCO
3
to 100 mL with 0.1 N NaOH (carbonate-
free) solution.
Statistical Analyses
Means and standard errors were obtained for each sam-
ple pair using Microsoft Excel Statistical Analysis Pack-
5
Microsoft Excel, Version 97, Microsoft Corp., Redmond, WA
98052-6399.
6
SAS software, Version 6.12, SAS Institute Inc., Cary, NC 27513.
age.
5
In addition, an analysis with SAS PROC GLM
6
eval-
uated the statistical equivalence of grouped means based
onsample size andaddedCO
2
solution. Interactioneffects
and non-normality concerns were also evaluated using
SAS software.
6
Statistical signicance and mean grouping
were based on P < 0.01.
RESULTS AND DISCUSSION
Tables 1 and 2 summarize the data for the egg albumen
and egg yolk samples with added CO
2
solution, respec-
tively. The CO
2
content (mg CO
2
/g sample) was obtained
by taking the predicted CO
2
concentration fromthe multi-
ple regression equation and subtracting the overall mean
weighted average CO
2
concentration (1.10 mg CO
2
/mL
solution) times the added CO
2
solution. This result was
then divided by the sample mass. No interaction effects or
non-normality effects were observed for yolk or albumen
data. These results indicate that CO
2
concentration within
the egg albumen samples, egg yolk samples, and CO
2
solutions were statistically equal with an overall mean
value of 1.66 mg/g in the albumen, 0.09 mg/g in the
yolk, and 1.10 g/mL in the CO
2
solutions, respectively.
These results are shown in Table 3. Pooled data compari-
sons found no statistical differences in CO
2
concentration
for different size samples; however, the standard error
generally decreased as sample size increased.
As sample size increases (more CO
2
in sample), the
inuence of backgroundCO
2
levels on measurements will
decrease. In our laboratory and pilot plant area, back-
ground CO
2
levels have been observed to range from0.04
to 0.5% (5,000 ppm). CO
2
is typically present in air at
approximately 0.04%. Thus, a 250-mL container (at 0.04%
CO
2
) should contain approximately 0.2 mg CO
2
. Our ex-
perience has indicated that background CO
2
levels can
vary considerably, upto 2 mg CO
2
, anda backgroundCO
2
measurement needs to be done for each set of samples.
Background CO
2
content was found to be slightly nega-
tive for data in Tables 1 and 2 and was positive for Table
4 data. Besides variations in background CO
2
levels, CO
2
measurements are subject to operator errors in accurately
determining titration endpoint, determining normality of
solutions, and reading burette volumes. These errors be-
come less signicant as sample size increases and, thus,
are the main factors in determining a recommended sam-
ple size.
The developed method accurately measured the
amount of CO
2
present in the egg albumen and egg yolk
samples for samples of 10 g or less. In addition, the CO
2
concentrations determined for the added CO
2
solution
was shown to be statistically equivalent between sample
groups. It was also observedthat standarderror increased
as sample size decreased below 6 g; therefore, the recom-
mended sample size would be 6-g samples. Overall, these
results show that this method will accurately measure
CO
2
concentration in egg albumen and yolk samples.
A follow-up study with fresh eggs was performed to
better determine method precision for 6-g samples. Two-
day-old eggs were obtained from Hy-Line chickens. The
KEENER ET AL. 986
TABLE 1. Summary of egg albumen CO
2
concentration data
1
Average albumen
CO
2
added Average CO
2
CO
2
concentration
Mass
2
(g) (mL) content
2
(mg) (mg/g albumen)
0.00 0.00 0.82 0.120 . . .
1.99 0.014 0.00 2.57 0.036 1.57 0.194
a
3.99 0.014 0.00 6.14 0.280 1.74 0.076
a
6.04 0.035 0.00 9.37 0.040 1.68 0.016
a
8.05 0.000 0.00 13.53 0.401 1.78 0.049
a
10.04 0.063 0.00 18.40 3.28 1.91 0.314
a
0.00 2.00 1.69 0.160 . . .
1.97 0.07 2.00 5.12 0.601 1.68 0.232
a
4.03 0.124 2.00 8.57 0.120 1.78 0.029
a
6.04 0.063 2.00 11.18 0.200 1.62 0.016
a
7.99 0.035 2.00 15.06 0.481 1.71 0.067
a
9.9 0.049 2.00 17.89 0.320 1.65 0.023
a
0.00 4.00 3.56 1.121 . . .
2.12 0.077 4.00 7.27 0.280 1.83 0.068
a
4.01 0.000 4.00 10.27 0.361 1.67 0.089
a
5.99 0.049 4.00 15.26 1.803 1.94 0.284
a
8.02 0.077 4.00 16.79 0.200 1.64 0.008
a
9.99 0.127 4.00 19.56 0.441 1.60 0.023
a
a
Letters indicate statistical equivalence at P < 0.01.
1
Average of two samples.
2
Means standard error.
TABLE 2. Summary of egg yolk CO
2
concentration data
1
Average yolk
CO
2
added Average CO
2
CO
2
concentration
Mass
2
(g) (mL) content
2
(mg) (mg/g yolk)
0.00 0.00 0.76 0.440 . . .
2.05 0.028 0.00 0.19 0.120 0.50 0.066
a
4.05 0.014 0.00 0.34 0.320 0.11 0.079
a
5.97 0.141 0.00 0.50 0.160 0.22 0.028
a
8.00 0.035 0.00 0.67 0.160 0.18 0.019
a
9.97 0.056 0.00 0.50 0.080 0.13 0.008
a
0.00 2.00 1.89 0.120 . . .
1.99 0.042 2.00 1.83 0.841 0.23 0.427
a
4.00 0.077 2.00 2.14 0.481 0.19 0.123
a
6.01 0.120 2.00 2.77 0.080 0.23 0.017
a
7.99 0.063 2.00 2.51 0.120 0.14 0.016
a
9.94 0.000 2.00 2.26 0.240 0.089 0.024
a
0.00 4.00 4.10 0.200 . . .
1.99 0.007 4.00 4.61 0.280 0.52 0.138
a
4.03 0.014 4.00 4.78 1.082 0.30 0.269
a
6.02 0.014 4.00 4.35 0.401 0.13 0.066
a
7.97 0.021 4.00 5.37 0.561 0.22 0.069
a
10.04 0.014 4.00 5.15 0.240 0.15 0.024
a
a
Letters indicate statistically equivalence at P < 0.01.
1
Average of two samples.
2
Means standard error.
TABLE 3. Average CO
2
concentrations determined for egg albumen, egg yolk, and CO
2
solution samples
CO
2
Concentration
1
CO
2
Solution (mg/mL) Albumen (mg/g) Yolk (mg/g)
Albumen + CO
2
1.01 0.14
a
1.66 0.03
Yolk + CO
2
1.11 0.11
a
. . . 0.09 0.02
CO
2
Solution 1.23 0.11
a
. . . . . .
CO
2
Solution 1.26 0.08
a
. . . . . .
Literature
2
. . . 1.90 0.00
a
Letters indicate statistical equivalence at P < 0.01.
1
Means standard error.
2
Fresh egg (Mueller, 1958).
CHEMICAL METHOD FOR DETERMINATION OF CARBON DIOXIDE IN EGGS 987
TABLE 4. CO
2
measurements of fresh egg samples
Coefcient of
CO
2
Content
1
CO
2
Concentration
1
variation
(mg) (mg/g) (%)
Albumen blank 0.938 0.033 . . . 3.5
Yolk blank 0.158 0.006 . . . 3.8
Albumen 8.64 0.0024 1.44 0.0004 0.028
Yolk 0.456 0.0000132 0.076 0.0000022 0.0029
1
Mean standard error.
eggs were pooled, and ve sample blanks, ve 6-g yolk
samples, and ve 6-g albumen samples were made. These
eggs were then tested using the prescribed method, and
the results are shown in Table 4. From these results, one
can observe that the CO
2
concentration in the fresh egg
albumen and egg yolk is 1.44 mg/g and 0.076 mg/g,
respectively. The CV for the albumen and yolk were 0.03
and 0.003%, respectively. These results suggest that using
ve 6-g sample averages will allow measurements to be
within 0.03% precision for albumen and 0.003% precision
for yolk. In practice, this method produced CO
2
measure-
ments in albumen and yolk with CV of 5% or less for 15
6-g samples. The large increase in sample size and CV
resulted from CO
2
content differences between individ-
ual eggs.
In conclusion, these results suggest that the chemical
method presented here can be accurately and precisely
used to measure CO
2
concentrations in egg albumen and
egg yolk samples. Emphasis should be placed on de-
termining the background CO
2
levels because they can
vary considerably with time. In our laboratory, this
method of CO
2
measurement with 15 6-g egg albumen
and egg yolk samples has consistently produced CV of
5% or less. This developed method for determining CO
2
content also has possibilities in other food systems be-
cause it has been shown to work in high protein (egg
albumen) and high lipid (egg yolk) products.
ACKNOWLEDGMENTS
The authors would like to acknowledge the assistance
of Roger Thompson of the USDA Fermentation Labora-
tory (NorthCarolina State University, Raleigh, North Car-
olina) who provided details on the method developed
for determining CO
2
content in cucumber brines.
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2
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