J. Dairy Sci. 89:859–861
 American Dairy Science Association, 2006.
Technical Note: Determination of Acidity in Whole Raw Milk:
Comparison of Results Obtained by Two Different Analytical Methods
M. A. Fabro,*1 H. V. Milanesio,* L. M. Robert,* J. L. Speranza,* M. Murphy,†
G. Rodrı´guez,† and R. Castan˜eda†
*Divisio´n Rafaela, Centro de Investigaciones Tecnolo´gicas de la Industria La´ctea, Instituto Nacional de Tecnologı´a Industrial,
Rafaela, 2300, Argentina
†Divisio´n Miguelete, Centro de Investigaciones Tecnolo´gicas de la Industria La´ctea, Instituto Nacional de Tecnologı´a Industrial,
General Paz and Alvarellos, San Martı´n, Buenos Aires, Argentina
ABSTRACT
In Argentina, one analytical method is usually car-
ried out to determine acidity in whole raw milk: the
Instituto Nacional de Racionalizacio´n de Materiales
standard (no. 14005), based on the Dornic method of
French origin. In a national and international regula-
tion, the Association of Official Analytical Chemists In-
ternational method (no. 947.05) is proposed as the stan-
dard method of analysis. Although these methods have
the same foundation, there is no evidence that results
obtained using the 2 methods are equivalent. The pres-
ence of some trends and discordant data lead us to
perform a statistical study to verify the equivalency of
the obtained results. We analyzed 266 samples and the
existence of significant differences between the results
obtained by both methods was determined.
Key words: acidity, milk, methodology
INTRODUCTION
What is usually known as milk acidity is the result
of titration (Alais, 1971). Titratable acidity is the capac-
ity of combination with a base (Goded y Mur, 1966).
The measurement principle is unique, and is based on
adding, to a given volume of milk, the necessary volume
of alkaline solution (sodium hydroxide) of an exact con-
centration until the neutralization point is reached,
which is determined by the presence of an indicator,
generally phenolphthalein, which turns from colorless
to pink at pH 8.4.
Although the measurement principle is the same,
there are variations among methods. Goded y Mur
(1966) differentiated the official methods of several
countries according to the concentration of the alkaline
solution, the milk volume to titrate, and the concentra-
tion of phenolphthalein. In addition to methodological
Received June 27, 2005.
Accepted August 29, 2005.
1Corresponding author: mfabro@inti.gov.ar
859
variations, there are different units in which to express
the obtained results: Dornic degree (°D), Soxhlet-
Henkel degrees (SH), and Thorner degrees.
These variations can cause possible incongruities in
the results obtained by one or other technique in the
same milk sample. Alais (1971) states that, although
the acidity measurement is simple to conduct, there
are several sources of error, including the amount of
indicator and the determination of the end-point of the
titration. Goded y Mur (1966) indicated as possible
sources of error the indicator selected and its concentra-
tion, the alkaline solution selected and its concentra-
tion, the titration speed, the working temperature, and
the milk dilution.
In the dairy laboratories of Argentina, determination
of milk acidity is usually made using the method of
Instituto Nacional de Racionalizacio´n de Materiales
(IRAM) standard 14005 (IRAM, 1976) because it is the
national standard method and it is somewhat faster to
perform than the alternative technique, method 947.05
of the Association of Official Analytical Chemists
(AOAC, 1990). Both methods are based upon the same
principle but there are differences in the volume and
dilution of the test sample and in the titration.
The AOAC method (no. 947.05; AOAC, 1990) was
established as a methodology of analysis for acidity
in a regulation: SGT No. 3/REC No. 40/94 of Co´digo
Alimentario Argentino Anexo Mercosur (Co´digo Ali-
mentario Argentino, 2003). According to this methodol-
ogy, certain values of acidity were established as toler-
ated limits for normal milk. Many milk samples that
were normal by the IRAM method were outside the
tolerated limits when analyzed using the AOAC
method. When comparing data among laboratories of
different companies and different countries using one
standard or another, some discordant data and trends
began to appear. In the course of our research, we found
out that these values of acidity were the normal limits
fixed for milk under another standard, IRAM 14017,
using the methodology of IRAM 14005 (IRAM, 1976).
Although the different methods described above have
the same foundation, there is no evidence that the re-

FABRO ET AL.860
sults obtained are equivalent. This observation led us
to perform a statistical study to verify the equivalency
in the obtained results using different standards, which
may help in the revision of the tolerated limits for
each methodology.
MATERIALS AND METHODS
To determine if differences existed between the re-
sults obtained by both methodologies, 266 samples were
analyzed by both techniques in the laboratories of the
Instituto Nacional de Tecnologı´a Industrial (Rafaela,
Argentina) to enable a statistical analysis of the col-
lected data.
Sample Characteristics
Our laboratory worked with 8 dairy companies lo-
cated in the main milk production areas of Argentina
to collect representative samples of Argentina’s milk
production. Whole raw milk samples were selected, ho-
mogeneously distributed by region and time over 4 mo
of sampling.
Sampling Procedure
The sampling and delivery of the samples was carried
out by the companies’ own transportation allowing the
samples to be in the laboratory within 16 h after sam-
pling. No preservative was used. The samples were
maintained from the time of sampling until the time
of analysis at temperatures between 2 and 10°C. Each
sample was analyzed using both methods within 10
min.
Analytical Methods
AOAC Method. To 20 mL of milk sample was added
40 mL of boiled and cooled distilled water, and 2 mL
of phenolphthalein (prepared at 1% in 95% ethanol).
The mixture was titrated with standardized 0.1 M
NaOH until the first color change (to pink) persisted
for 30 s. One more drop of 0.1 M NaOH was added and
the final volume of 0.1 M NaOH added was noted.
IRAM14005 Method.To 10 mL of sample was added
3 drops of phenolphthalein solution (prepared at 2% in
96% ethanol). The mixture was titrated with standard-
ized 0.111N NaOH until the first color change (to pink)
persisted for 30 s. One more drop of 0.111N NaOH was
added and the final volume of 0.111 N NaOH added
was noted.
The same formula was used for both methods to ex-
press the results in equivalent format or units:
Journal of Dairy Science Vol. 89 No. 3, 2006
Table 1. Results obtained for determination of titratable acidity in
256 milk samples using 2 methods. All values are expressed in milli-
grams of lactic acid /100 mL of milk
Method1
AOAC IRAM Difference
Average 117.581132 149.664151 32.083019
Standard deviation 14.0266395 11.4140363 18.454079
1AOAC = Method 947.05, Association of Official Analytical Chem-
ists (AOAC, 1990); IRAM = method 14005, Instituto de Racionaliza-
cio´n de Materiales (IRAM, 1976).
Lactic acid (mg)/100 mL of milk =
(Vg × N × 90 × 100)/Vm
where Vg = volume of NaOH solution added, N = concen-
tration of sodium hydroxide standardized solution ex-
pressed in Eq/L, 90 = equivalent weight of lactic acid,
and Vm = volume of milk used for titration.
Statistical Methods
Two statistical tests to compare the methods were
applied: pair comparison design and single-factor vari-
ance analysis (Montgomery, 1991).
RESULTS AND DISCUSSION
A total of 566 analytical results were obtained from
266 samples; 265 samples were analyzed by both meth-
ods and 1 sample was analyzed 18 times by both
methods.
Statistical Tests
The pair comparison design was used to analyze the
530 results from 265 samples analyzed by both meth-
ods, and the single-factor variance test was used for
the results from 1 sample analyzed 18 times by both
methods.
Pair ComparisonDesign.To prove the Ho hypothe-
sis: 1 = 2 is equivalent it was necessary to prove Ho:
d = 0. The statistical test for those hypotheses was to.
In this case, to = 28.301261 and t∝/2,n−1 = t0.025.264 =
1.960. As to > t∝/2,n−1, the average of the differences is
not null; for a 95% confidence level (α = 0.05), there is
a statistically significant difference between both meth-
ods. Some parameters (in mg of lactic acid/100 mL of
milk) of the obtained results from 256 samples analyzed
using both methods are shown in Table 1.
Variance Analysis. Table 2 shows the variance
analysis of the 36 results obtained from analysis of 1
sample multiple times using both methods. With a 95%
confidence level (α = 0.05), it is possible to say that the

TECHNICAL NOTE: DETERMINATION OF ACIDITY IN RAW MILK 861
Table 2. Variance analysis of 36 determinations of titratable acidity (18 for each method) in 1 milk sample using 2 methods
Method1 Acidity (yi; mg of lactic acid/100 mL of milk) Σyi Mean yi
AOAC 111-112-110-110-110-112-110-110-111-112-111-112-111-112-112-110-110-111 1,997 110.94
IRAM 149-152-158-153-153-153-151-149-149-146-141-154-151-141-148-143-159-143 2,693 149.61
Mean
Source of variation Sum of squares2 df square3 F0 =
Mean Square Treatments
Mean Square Experimental
Treatments4 13,456.002 1 13,456.002 939
Experimental5 487.220 34 14.330
Error total6 13,943.222 35
F7α,a−1,n−1 = F0.05, 1, 34 = 4.12
Fo >>>F0.05, 1, 34
1AOAC = Method 947.05, Association of Official Analytical Chemists (AOAC, 1990); IRAM = method 14005, Instituto de Racionalizacio´n
de Materiales (IRAM, 1976).
2Sum of square (Montgomery, 1991).
3Mean square = sum of square/degrees of freedom.
4Variation between values of both methodologies.
5Variation between values from the same methodology.
6Total variability between the all values (yi).
7Value of the distribution F for these test conditions and with 95% confidence.
use of one technique or another significantly influenced
the result of the acidity expressed in milligrams of lactic
acid per 100 mL of milk.
DISCUSSION
After applying 2 statistical tests, it was concluded
that there are statistically significant differences (with
a 95% confidence level) between the analytical results
obtained by the 2 methods. The main causes of the
differences could be the larger sample size and the sam-
ple dilution in the AOAC method. Both factors would
allow better and earlier observation of the color change
to pink, and thus addition of NaOH would stop earlier.
The resultant acidity value would be lower. The differ-
ence in concentration of the alkaline solutions is very
small and would not be an important factor.
CONCLUSIONS
The results of this study allow us to conclude that
differences exist in the results obtained by both method-
ologies applied on the same milk sample. Results ob-
tained using one method are not equivalent to those
obtained by the other method, which correlates with
anecdotal reports from dairy industry laboratories. The
evidence obtained as a result of this work would allow
Journal of Dairy Science Vol. 89 No. 3, 2006
us to avoid errors that could be present when results
obtained by diverse methodologies are compared.
The results of this study are important for the Argen-
tinean dairy industry. With this information, the indus-
try can ask for a revision of the tolerated limit for acidity
in the resolution SGT No. 3/REC No. 40/94 of Co´digo
Alimentario Argentino Anexo Mercosur.
ACKNOWLEDGMENTS
The authors acknowledge the collaboration of the par-
ticipating dairy industries and their personnel and of
all the personnel of INTI La´cteos, especially Eduardo
Storani for his collaboration with the translation.
REFERENCES
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AOAC. 1990. Official Methods of Analysis. 15th ed. Association of
Official Analytical Chemists, Arlington, VA.
Co´digo Alimentario Argentino. 2003. De la Canal & Asociados SRL,
Olivos, Buenos Aires, Argentina.
Goded y Mur, A. 1966. Te´cnicas modernas aplicadas al ana´lisis de
la leche. Pages 62–64 in ciones Fı´sicas.1st ed. Dossat, Madrid,
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Instituto Nacional de Racionalizacio´n de Materiales. 1976. Standard
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Montgomery, D. C. 1991. Disen˜o y ana´lisis de experimentos. Grupo
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