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Food Anal.

Methods (2016) 9:11501154


DOI 10.1007/s12161-015-0294-4

Simplifying Iron Determination with o-Phenanthroline in Food


Ashes Using 2-Nitrophenol as an Acid-Base Indicator
P. Serra-Mora 1 & Y. Moliner-Martnez 1 & R. Herrez-Hernndez 1 & J. Verd-Andrs 1 &
P. Campns-Falc 1

Received: 27 February 2015 / Accepted: 19 August 2015 / Published online: 28 August 2015
# Springer Science+Business Media New York 2015

Abstract Iron determination in foods can be performed by various tissues (Food and Nutrition Division FAO 2001). For
measuring the absorbance of the complex formed between this these reasons, the determination of iron in food is a need, and
element and o-phenanthroline, after dry mineralization of the several national and international norms describe standardized
sample and treatment with HCl 6 M. An experimental prob- methods for its quantification in different kinds of matrices.
lem is how to adjust the pH of the measuring solution to the Most of these methods are based on the dry mineralization
optimal value of 3.5. In official norms, a preliminary test to of the sample (ISO 55161978), solubilization of the ashes,
determine the volume of acetate buffer solution needed for and iron determination in the resulting solution, measuring the
each sample is required, which makes the method unsuitable absorbance of the complex formed between iron and o-
for routine analysis. In this work, 2-nitrophenol is proposed as phenanthroline (ISO 5517:1978; NTE INEN 0400 1979;
an acid-base indicator for neutralizing in situ each sample, COVENIN 117083; Method 937.03 1990; NMX-F-503-
thus avoiding the need of preliminary tests. As a result, the SCFI-2011). This method has similar analytical properties
experimental procedure is simplified, and less sample, mate- than the iron determination by atomic absorption spectrome-
rials, time, and experimental effort are needed. Reproducibil- try, and the results obtained are statistically comparable (Tee
ity (lower than 5 % in triplicate for four different samples) was et al. 1989; Hosseinimehr et al. 2007).
in agreement with the quality standards required at the con- Although iron-o-phenanthroline complex coloration de-
centration level of iron found in foods, and the recovery ((101 velops at a pH between 2 and 9, its intensity is constant only
5)%, n=8) is consistent with the values found in the bibli- between pHs 3.5 and 4.5 (ISO 5517:1978). The main exper-
ography for this determination. imental problem of the official procedures is how to adjust the
pH of the measuring solution to the optimal value. Because of
Keywords Iron . Visible spectroscopy . Food ash the high acidity of the solution obtained from the ashes, a
preliminary test is carried out to determine the volume of
acetate buffer needed for each sample, so a titration of an
aliquot of each sample solution is required; the obtained vol-
Introduction
ume is then added to other sample aliquot which is then treated
for absorbance measurement (ISO 5517:1978; NTE INEN
Iron has several vital functions in the body. It serves as a
0400 1979; COVENIN 117083; Method 937.03 1990).
carrier of oxygen from the lungs to the tissues by red blood
These operations increase the amount of sample and materials
cell hemoglobin, as a transport medium for electrons within
required, as well as the time and experimental effort necessary
cells, and as an integrated part of important enzyme systems in
to perform the determination, making this method unsuitable
for routine purposes.
* J. Verd-Andrs In the present brief, the use of 2-nitrophenol as an acid-base
jorge.verdu@uv.es indicator to neutralize in situ the sample is proposed, thus
avoiding the need of a preliminary titration. 2-Nitrophenol
1
Departament de Qumica Analtica, Universitat de Valncia, c/Dr. presents a change of color interval between pHs 5.0 and 7.0
Moliner 50, 46100 Burjassot, Valencia, Spain (Sabnis 2007), being colorless at acidic conditions and yellow
Food Anal. Methods (2016) 9:11501154 1151

at neutral-basic pHs. For this reason, at pH=3.5, it does not volume of sodium acetate buffer determined in the previous
interfere the spectroscopic determination of iron. step (for the same volume of iron solution), 2 mL of 0.25 % o-
phenanthroline, and water up to the mark. After 15 min, the
developed color was measured against water blank at 510 nm,
Materials and Methods correcting the signal by subtracting the absorbance value at
700 nm.
Materials and Instruments In the in situ 2-nitrophenol pH adjustment procedure, iron
calibration solutions were obtained by placing in 50-mL vol-
All reagents used were of analytical grade. Preparation details umetric flasks in this order (mixing after each addition) ali-
can be found elsewhere (ISO 5517:1978). quots of the 20 mg/L iron solution (010 mL), water until an
Iron stock solution of iron at a concentration of 200 mg/L approximate total volume of 20 mL, 1 mL of 1 % ascorbic
was prepared by dissolving iron (II) and ammonium sulfate acid, and one drop of the solution of 2-nitrophenol. Next,
hexahydrate, Fe(NH4)2(SO4)26H2O (Prolabo, Fontenay- NaOH 2 M was added drop by drop until the resulting solution
sous-Bois, France) in water, adding 1 mL of concentrated became yellow. Then, HCl 1 M was added drop by drop until
HCl (37 %, 0.00001 % maximum content in iron, from the solution became colorless. In such a way, the pH of the
Scharlau, Barcelona, Spain) for each 100 mL of solution pre- solution was slightly acidic. Next, 5 mL of 2 % citrate buffer
pared. A working solution of 20 mg/L was prepared by dilu- pH=3.5 was added to adjust the optimum pH, followed by
tion of the concentrated standard with water, and 1 mL of 2 mL of 0.25 % o-phenanthroline. Finally, water was added up
concentrated HCl was added for each 100 mL of diluted so- to the mark. After 15 min, the developed color was measured
lution. 0.25 % o-phenanthroline aqueous solution was pre- against water blank at 510 nm, correcting the signal by
pared by dissolving o-phenanthroline hydrochloride subtracting the absorbance value at 700 nm.
monohydrate (Sigma-Aldrich, Steinheim, Germany) in water. Four kinds of samples were employed throughout the
2 % citric/citrate buffer (pH=3.5) was prepared by dissolving study: corn flakes, natural roasted grinded coffee, a cocoa
citric acid monohydrate (Panreac, Barcelona, Spain) in water preparation in powder, and pork liver pate (22.5 % pork liver).
and adjusting the pH with 2 M NaOH (J.T. Baker, Deventer, All samples were acquired in local supermarkets. According
Holland). 1 % ascorbic acid aqueous solution was prepared by to the labels, cocoa preparation and corn flakes contained
dissolving ascorbic acid (VWR Prolabo, Leuven, Belgium) in added iron. Samples were directly analyzed, except corn
water; ascorbic acid was preferred over hydroquinone or flakes that were ground before analysis.
hydroxylammonium chloride because of its lower toxicity. Each sample was analyzed in triplicate by both methodol-
0.3 % solution of 2-nitrophenol was prepared by dissolving ogies. In addition, two portions of each sample were fortified
the pure reagent (Merck Schuchardt, Germany) in ethanol- with known amounts of iron in order to perform recovery
water (1:1); ethanol was purchased from Romil (Cambridge, studies.
England). Sodium acetate solution at a concentration of 450 g/ For analysis, portions of the samples (about 10 g) were
L was prepared by dissolving sodium acetate anhydrous accurately weighted in dry porcelain crucibles and left over-
(Merck, Darmstadt, Germany) in water. night (12 h) in a muffle furnace at 550 C, until a whitish or
For pH measurements, a Crison system (Barcelona, Spain) greyish ash was obtained. For the recovery studies, the
was used. amount of samples was 5 g, and about 200 g of iron was
For absorbance measurements, a diode-array spectropho- added (1 mL of the 200 mg/L iron standard).
tometer (Agilent 8453, Santa Clara, USA) was used. As any carbon residue was formed, ashes were treated by
slowly adding 10 mL of HCl 6 M into the porcelain crucible.
Methods The resulting mixture was heated during 30 min until slow
boiling to hydrolyze pyrophosphates to orthophosphates, in
Solutions for iron calibration by the acetate titration method- order to avoid interferences in the iron determination, and
ology were obtained by placing in small Erlenmeyer flasks the water was periodically added to maintain the initial volume.
desired volume of 20 mg/L standard solution (010 mL), After this treatment, the content of the crucibles was trans-
1 mL of 1 % ascorbic acid and water up to an approximate ferred quantitatively to 100-mL volumetric flasks, and water
total volume of 20 mL; this mixture was then titrated potenti- was added up to the mark.
ometrically with the sodium acetate solution until a pH value For iron determination in the resulting sample solutions,
of 3.5 was reached. These values were registered as they have the same procedures described for the calibration solutions
to be used in the next step. In 50-mL volumetric flasks were were applied, but replacing the iron standard solution by
placed in this order and mixed after each addition the same 10 mL of the solution obtained after sample treatment. Owing
volumes of 20 mg/L iron solution tested before, water up to an to the higher acidity of the solutions obtained from the sam-
approximate volume of 20 mL, 1 mL of 1 % ascorbic acid, the ples, the amounts of sodium acetate or NaOH 2 M required to
1152 Food Anal. Methods (2016) 9:11501154

Table 1 Calibration lines obtained by the two tested methodologies

Method Fe concentration interval (mg/L) asa bsb r syx Number of data points

Sodium acetate titration 04.6 (3.01.1)103 0.19690.0004 0.999986 0.0019 8


In situ 2-nitrophenol pH adjustment 04.6 (4.40.7)103 0.19630.0002 0.999994 0.0012 8

neutralize them were higher than those needed for the calibra- by applying t test for two means of equal variance, except for
tion solutions. coffee samples; for the later sample, the t test for two means of
Samples were also analyzed by the standard additions not equal variance was employed (Miller and Miller 2000).
method using the in situ 2-nitrophenol pH adjustment method. Moreover, both methodologies lead to equivalent iron
In this case, 5 mL of the sample solutions was employed, and contents.
06 mL (for the corn flakes and cocoa preparation samples) or The iron content found in the corn flake sample was statis-
07 mL (coffee or pate samples) of 20 mg/L of iron standard tically equivalent (P=0.95) to the value declared in its label
solutions were introduced into six different 50-mL volumetric (70 mg/kg). However, the result found for cocoa preparation
flasks. Then, the mixtures were processed by in situ 2- was statistically lower (P= 0.95) than the declared value
nitrophenol pH adjustment method under the conditions de- (147 mg/kg). Iron contents obtained for the coffee sample
scribed before. were consistent with the values typically found in this kind
of samples. For example, iron concentrations ranging from
12.0 to 617.0 mg/kg have been found in roasted and ground
Results and Discussion coffees (Pohl et al. 2013). Finally, the iron content found in
pork liver pate was also consistent with previously reported
Iron Content in the Samples values (55 mg/kg for canned, not specified, liver pate (USDA
National Nutrient Database for Standard Reference 2011)).
The iron calibration results obtained with the two procedures The repeatability of the results was suitable according to
described above (sodium acetate titration and in situ 2- the quality standards required at these concentration levels.
nitrophenol pH adjustment) are listed in Table 1. The linearity For example, European legislation for the analysis of feed
holds true until, at least, an iron concentration of 4.6 mg/L, samples (Commission Regulation (EC) No 152/2009) estab-
with estimated detection limits of 0.020.03 mg/L. A statisti- lishes that the difference between the results of two parallel
cal test for coincidence of lines by using a dummy variable determinations carried out on the same sample by the same
was performed (Andrade-Garda et al. 2013). For a 95 % con- analyst must not exceed 5 mg/kg, in absolute value, for con-
fidence interval, 2 and 3 coefficients were statistically null tents of the trace element concerned up to 50 mg/kg, 10 % of
(data not shown), which indicated that both lines are identical the higher result for contents of the trace element concerned
(same intercepts and slopes); thus, both methodologies lead to from 50 up to 100 mg/kg, 10 mg/kg, in absolute value, for
equivalent calibration parameters. contents of the trace element concerned from 100 up to
The iron content values obtained for the four samples 200 mg/kg, or 5 % of the higher result for contents of the trace
assayed by the sodium acetate titration and in situ 2- element concerned above 200 mg/kg.
nitrophenol pH adjustment methods are shown in Table 2. Taking these legislated values as a reference, and using the
As it can be seen, the results obtained by both procedures worst duplicate for each sample (it should be noted that since
are not significantly different for a 95 % confidence interval each sample was analyzed in triplicate, three different

Table 2 Results obtained for iron content in the four samples assayed by the two tested methods

Sample Sodium acetate titration method In situ 2-nitrophenol pH adjustment method Equal values
(t test, P=0.05)
Fe (mg/kg) (means) Maximum difference Fe (mg/kg) (means) Maximum difference
(mg/kg) (absolute) (mg/kg) (mg/kg) (absolute) (mg/kg)

Corn flakes 69.2; 69.0; 68.4 68.80.4 0.8 68.5; 68.9; 68.1 68.50.4 0.8 Yes
Coffee 57.7; 55.6; 58.8 57.41.6 3.2 58.1; 54.9; 59.6 57.52.4 4.1 Yes
Cocoa preparation 140.6; 141.2; 139.0 140.31.1 2.2 139.9; 140.6; 138.6 139.71.0 2.0 Yes
Pate 44.0; 47.1; 44.1 45.11.8 3.2 44.2; 46.3; 44.0 44.81.3 2.3 yes
Food Anal. Methods (2016) 9:11501154 1153

Table 3 Recovery results obtained for the four samples by the in situ 2- Table 5 Iron contents found in the samples assayed by the standard
nitrophenol pH adjustment method additions and in situ 2-nitrophenol pH adjustment method and in com-
parison with those obtained by using external calibration (see Table 2)
Sample Fe added (g) Fe found (g) % Recovery
Sample (means) (mg/kg) Equal values (t test, P=0.05)
Corn flakes 232 225, 254 97, 109
Coffee 232 229, 237 98, 102 Corn flakes 69.50.8 Yes
Cocoa preparation 232 234, 247 101, 106 Coffee 57.40.7 Yes
Pork liver pate 232 231, 223 100, 96 Cocoa preparation 141.81.8 Yes
Global (means) (1015)% Pate 45.80.7 Yes

duplicated values can be chosen), the repeatability for the in of its higher buffer capacity at pH=3.5 (pKa1 =3.13; pKa2 =
situ 2-nitrophenol pH adjustment was 0.8 mg/kg for corn 4.76; pKa3 =6.4 (Budavari 1989)) compared to that of the
flakes (allowed, 6.9 mg/kg), 4.1 mg/kg for coffee (allowed, acetic/acetate buffer (pKa =4.74 (Budavari 1989)). Then, the
6.0 mg/kg), 2.0 mg/kg for cocoa preparation (allowed, proposed methodology is a better alternative for adjusting the
10 mg/kg), and 2.3 mg/kg for pork liver pate (allowed, pH.
5 mg/kg). Therefore, the repeatability was satisfactory, as even
in the worst case it was under the 68 % of the allowed value.
Study of Matrix Effect
The repeatability is rather similar to that achieved by the
sodium acetate titration approach: 0.8 mg/kg for corn flakes
In order to check whether the determination was affected by
(allowed, 6.9 mg/kg), 3.2 mg/kg for coffee (allowed,
matrix effect, the standard addition method was employed to
5.7 mg/kg), 2.2 mg/kg for cocoa preparation (allowed,
determine iron content in the four samples assayed (see
10 mg/kg), and 3.2 mg/kg for pork liver pate (allowed,
Table 4). For a 95 % confidence interval, 3 coefficients were
5 mg/kg). For this method, even in the worst case, the repeat-
statistically null for all the standard addition lines compared to
ability was under the 64 % of the acceptable value.
the calibration line shown in Table 1 (Andrade-Garda et al.
The recovery obtained by the proposed in situ 2-
2013, data not shown). This means that the slopes of the stan-
nitrophenol pH adjustment method was also satisfactory.
dard addition lines were equivalent to the slope of the iron
The mean recovery obtained by analyzing each sample in
calibration line; therefore, it was concluded that matrix effect
duplicate was (1015)%, (n=8) as listed in Table 3. This
was absent. With the proposed methodology, the amounts of
value was statistically equivalent to 100 %; it was comparable
iron found with standard additions method were statistically
to other values published for iron determination in similar
equivalent to those found by using external calibration, with a
matrices, for example that obtained by Tee et al. (1989), which
confidence level of 95 % (Table 5).
was (9111)% (n=10).

pH Study Conclusions

The pH of the sample solutions was measured after the absor- It has been demonstrated that for iron determination with o-
bance measurements. The mean pH values obtained were phenanthroline in food ashes, the in situ neutralization using
(3.470.07) and (3.830.35), for all samples analyzed by 2-nitrophenol as an acid-base indicator is a faster and simpler
the in situ 2-nitrophenol pH adjustment and the sodium acetate alternative than the titration of a sample replicate with acetate
titration methods, respectively (n=12). This means that the buffer, as proposed in official norms. The proposed method
combined use of in situ pH adjustment to 44.5 and the addi- also leads to a better pH adjustment in the solution to be used
tion of citric/citrate buffer is a better option, probably because for absorbance measurement. The results obtained by the

Table 4 Application of the standard additions method for iron determination by the in situ 2-nitrophenol pH adjustment method

Sample Fe added concentration interval (mg/L) asa bsb r syx Number of data points

Corn flakes 02.8 0.27940.0014 0.19440.0009 0.99996 0.002 6


Coffee 03.25 0.21330.0012 0.19700.0006 0.99998 0.0017 6
Cocoa preparation 02.8 0.5580.002 0.19680.0014 0.99990 0.003 6
Pork liver pate 03.25 0.18780.0015 0.19800.0007 0.99997 0.002 6
1154 Food Anal. Methods (2016) 9:11501154

proposed methodology are equivalent to those obtained by the COVENIN 117083 (1983) Norma venezolana. Alimentos.
Determinacin de hierro (1 revisin). Caracas, Venezuela
reference method in terms of accuracy and repeatability. The
Food and Nutrition Division FAO (2001) Human vitamin and mineral
recovery obtained was (1015)%. These results fulfill the requirements. Report of a joint FAO/WHO expert consultation,
analytical requirements for this kind of analysis. In addition, Bangkok, Thailand. Rome
the determination is not affected by matrix effect. The pro- Hosseinimehr SJ, Bagheri G, Gholipoor M, Mokarami H (2007)
posed method can be considered a simplification of the refer- Comparative spectrophotometric and atomic absorption determina-
tion of iron content in wheat flour. J Biol Sci 7(2):451453
ence procedure; the determination of iron can be carried out in
ISO 55161978 (1978) Fruits, vegetables and derived products
an easier and faster way. Therefore, it can be considered a decomposition of organic matter prior to analysisashing
reliable alternative to the reference method. method. International Organization for Standardization,
Switzerland
Acknowledgments The authors are grateful to the Generalitat ISO 5517:1978 (1978) Fruit and vegetable productsdetermination
Valenciana (project GVACOMP/2013/155) for the financial support of iron content1,10-phenanthroline photometric method.
received. International Organization for Standardization, Switzerland
Method 937.03 (1990) Iron in plants. Colorimetric method. Official
Conflict of Interest P. Serra-Mora declares that there is no conflict of methods of analysis. Association of Official Analytical Chemists.
interest. Y. Moliner-Martnez declares that there is no conflict of interest. 15th Edition
R. Herrez-Hernndez declares that there is no conflict of interest. J. Miller JN, Miller JC (2000) Statistics and chemometrics for analytical
Verd-Andrs declares that there is no conflict of interest. P. Campns- chemistry, 4th edn. Pearson Education, England
Falc declares that there is no conflict of interest. This article does not NMX-F-503-SCFI-2011 (2011) Norma mexicana. Determinacin de
contain any studies with human or animal subjects. fierro en muestras de azcares. Direccin General de Normas.
Mxico
NTE INEN 0400 (1979) Conservas vegetales. Determinacin del
contenido de hierro. Norma Tcnica Ecuatoriana. Instituto
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