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Ascorbimetry: The microdetermination of ferric iron content of whole blood
ANALYTICAL
47,
BIOCHEMISTRY
123-131
(1972)
Ascorbimetry: The Microdetermination
of Ferric
M. Z. BARAKAT, Biochemistry
Iron Content
M. BASSIOUNI, F. EL-EINANY
Department, Madina
Faculty Nasr,
Received
A. S. FAYZALLA,
of Medicine, Cairo, Egypt
August
of Whole
Azhar
Blood AND
University,
8, 1971
In 1964 a method was reported (3) for the microdetermination of ferric iron with standard L-ascorbic acid using ammonium thiocyanate indicator solution. Although the method is accurate and sensitive, its use is limited to the determination of ferric iron in pharmaceutical preparations. The present work describes a modification for determining the ferric iron content of whole blood. MATERIALS
AND
METHODS
A total of sixty samples of normal adult sheep and buffalo blood was analyzed for ferric iron content. Of these, thirty samples were withdrawn from normal males and thirty samples from normal females of each species. The age of the animals ranged from 2 to 5 years. Equipment
and Reagents
1. Microburet of 5 ml capacity graduated to 0.01 ml. 2. Graduated pipets of 1, 2, 5, and 10 ml capacity. 3. Volumetric flasks of 10 ml capacity. 4. Erlenmeyer flasks of 50 and 100 ml capacity. 5. Standard L-ascorbic acid, e.g., 0.02 X, freshly prepared aqueous solution. 6. Standard N-bromosuccinimide (NBS), freshly prepared aqueous solution, e.g., 0.02 N solution. 7. Dilute sulfuric acid, 10% (v/v). 8. Dilute hydrochloric acid, 10% (v/v). 9. Methyl red indicator solution, 0.04% (w/v), alcoholic solution. 10. Standard titanous chloride, e.g., 0.01 N solution. 123 @ 1972 by
Academic
Press,
Inc.
124 I’alidity
of the Keclctiou
for
Quantitntive
Determination
Eeforc the reaction hctwvcn bascorhic acid and ferric iron was applied for the dctcrminatioii of the ferric iron content of blood, it was verified quantitatively. An accurately mcasurcd volume, e.g., 5 ml of a solution containing 0.9644 gm (I mmolc) of ferric ammonium sulfate per 100 ml was placed in a 100 ml stoppercd I~~rlcnmcycr flask and treated with an equal volume of dilute hydrochloric acid. Then an excess known volume of an aqueous solution containing 0.176 gm (1 mmole) of L-ascorbic acid per 100 ml was added. The mist,ure was thoroughly shaken and 0.1 ml of methyl red indicator solution was added. The excess of standard L-ascorbic acid was dctormincd by hack-titration with a standard X-bromosuccinimide solution containing 0.178 gm (1 mmolc) per 100 ml. The standard :V-bromosuccinimide solution was added gradually from a microburet with continuous shaking until the red color of the indicator was just dischargecl. The volume of standard L-ascorbic acid solution equivalent to the volume of ferric ammonium sulfate solution was easily calculated hy subtracting the volume of the ,V-hromosuccinimide tit’er from the volume of L-ascorbic acid added. The reaction was found to he stoichiometric at room temperature and the results were as follows: Vol.
ferric
ammonimn
10 Vol.
L-ascorbic
acid
4
soln.
(1 mmolr/lOO
acid
10.05
soln.
added
(1 mmolc/lOO
ml)
3.97
takrn
4.03
I
(ml)
(A) :
used
(ml)
(ml)
(A - B) :
4
2.98 ml)
(ml): 2
6
(1 mmole/lOO
4.98
taken
3
8 soln.
ml)
ml)
5 (02
L-ascorbic
(1 mmole/lOO
5
N-bromosuccinimide 9.95
Vol.
soln.
10
20 Titer
sulfate
2 (B)
:
2.04
3.02
1.02
1.96
0.98
A similar series of experiments was done using a known volume of ferric ammonium sulfate solution containing double the number of molecules of solute (2 mmoles). An excess known volume of L-ascorbic acid solution (1 mmole) per 100 ml was added. It was found that the reaction was stoichiometric at room temperature. The results were: Vol.
ferric
ammonium
10 Vol.
L-ascorbic 30
acid
sulfatcl
soln.
(2 mmoles/lOO
5
4
soln.
(1 mmole/lOO
15
12
ml) 3
ml)
added 9
taken
(ml) 2
(ml)
: 1
(A) : 6
3
FERRIC
Titer
N-bromosuccinimide 9.Y8
IRON
WHOLE
soln. (1 mmole/lOO
4.YX
4
Vol. L-ascorbic acid soln. (1 mmole/100
20.02
Ih-
10.02
125
BLOOD
ml) used (ml)
2.0;2
3.0" ml) taken (ml)
0.96
(A - B) :
5.YH
8
(B) :
3.98
2.02
Procedure
To an accurately measured volume, e.g., 5 ml of an unknown aqueous ferric iron solution in a 100 ml stoppered Erlenmeyer flask, add an equal volume of dilute hydrochloric acid and a double volume of a standard L-ascorbic acid solution. The standard ascorbic acid solution must be in excess. Shake well the contents of the flask for a period of 2 min. Then add 0.1 ml of methyl red indicator and titrate back the excess of standard L-ascorbic acid solution with standard N-bromosuccinimide solution of the same normality added gradually from a microburet, with shaking after each addition until the red color is just discharged. Carry out a blank experiment simultaneously and subtract the reading from the titer before calculation. Calculate the ferric iron content of the unknown solution from the expression as follows : ferric iron content (mg or pg) = V X C X
55.84 x 2 176
1’ = volume of standard L-ascorbic acid solution taken in the reaction and C = concentration of L-ascorbic acid in mg or pg/ml solution. RESULTS
Microdetemination
Recovery of Ferric Iron
A stock aqueous solution containing 0.1 gm/lOO ml ferric iron was prepared by dissolving 0.8635 gm ferric ammonium sulfate in distilled water, and the volume was made up with distilled water to 100 ml in a volumetric flask. The ferric iron content was determined by the proposed method in various known volumes of the st,ock solution as if they were unknowns, using an excess of 0.02 N L-ascorbic acid solution for reduction of ferric ions and 0.02 N N-bromosuccinimide solution for back-titration of excess L-ascorbic acid. The ferric iron content of a IO-fold diluted solution was also determined in various known volumes as if they were unknowns. The results are shown in Table 1.
126
BARAKAT
Recovery
of Ferric
ET
AL.
TABLE 1 Iron by the Proposed
Method
o.oa Nn Ferric iron bd
0.02 N GAscorbic acid added (ml)
10 9 8 7 6 5 4 3 2 1
(A) 12 11 10 9 8 7 6 5 4 3
(ml) (A - R
Ferric iron found (me)
8.98 7.98 7.18 6.24 5.36 4.57 3.58 2.72 1.78 0.90
10.03 8.91 8.02 6.97 5.99 5.10 4.00 3.04 1.99 1.01
0.004 N (ml)
0.004 Nb (ml)
(Pd
5.44 4.95 4.40 3.90 3.32 2.80 2.20 1.66 1.10 0.56 0.78
4.56 4.05 3.60 3.10 2.68 2.20 1.80 1.34 0.90 0.44 0.22
0.02 N N-Bromosuccinimide used (ml) (B) 3.02 3.02 2.82 2.76 2.64 2.43 2.42 2.28 2.22 2.10 Dilution
(Pd
0.004 N (ml)
1000 900 800 700 600 500 400 300 200 100 50
10 9 8 7 6 5 4 3 2 1 1
a 1 ml 0.02 N Lascorbic b 1 ml 0.004 N n-ascorbic
Comparative
L-Ascorbic acid taken
Error (%) 0.30 1.00 0.25 0.43 0.17 2.00 1.33 0.50 1.00
1 X 10
acid = 1.1168 mg ferric acid = 223.36 pg ferric
1019 905 804 692 599 491 402 299 201 98 49
1.90 0.56 0.50 1.14 0.17 1.80 0.50 0.33 0.50 2.00 2.00
iron. iron.
Analysis of Ferric Iron by the Proposed and the Previ0.u Ascorbimetric Method
Method
The proposed method was compared with the previous ascorbimetric method (3) using ammonium thiocyanate as an indicator for determining quantities of ferric iron varying from 500 to 50 pg as if they were unknowns. A known volume of 0.004 N L-ascorbic acid solution was added in excess and 0.004 N N-bromosuccinimide solution was used for back-titration using methyl red indicator. The results are listed in Table 2.
FERRIC
Comparative
Fe+++ (/.G) 500 400 300 200 100
50
IRON
IN
WHOLE
127
BLOOD
TABLE 2 Analysis of Ferric Iron by Proposed Method Previous Ascorbimetric Method Fe+++ found by proposed method (&) 496 402 299 201 9s 49
Interfering
and
Error (%I
Fe+++ found by previous method (hcd
Error (%I
o.so 0.50 0.33 0.50 2.00 2.00
500 39s 302 204 101 49
0.50 0.67 2.00 1.00 2.00
Substances
Typical interfering ions include silver, mercuric, and cupric ions. The silver cations may be separated by dilute HCI in the form of insoluble silver chloride and removed by filtration. The mercuric ions may be converted into nonionizable soluble potassium mercuric iodide (K?HgI,) with an excess of potassium iodide. Ferric ions and cupric ions will liberate an equivalent amount of iodine. which can be titrated with standard L-ascorbic acid solution using starch as an indicator (A). Sodium fluoride can be used to mask ferric ions in the presence of a few drops of ammonium thiocyanate solution until the red color disappears (6). Cupric ions wit,11 excess potassium iodide form cupric iodide, which decomposes spontaneously to cuprous iodide and free iodine, which can be titrated with the same standard L-ascorbic acid using starch indicator (B). The difference between the two titers (A B) corresponds to the ferric iron in the volume taken. Experimental
Error
The experimental error of the proposed method does not exceed ,270 when determining amounts varying from 10 mg to 50 /kg of ferric iron (Tables 1 and 2). Application
of the Proposed Method
(A) Determination of fewic iron content of ferric ammonium citrate. An accurately weighed amount of ferric ammonium citrate (in form of scales), e.g., 1 gm was brought into aqueous solution and the volume was completed with distilled water to 100 ml in a volumetric flask, so that 1 ml of the solution was equivalent to 10 mg ferric ammonium citrate.
128
BARAKAT
Recovery
of Ferric Iron Proposed Method Proposed
Ferric ammonium citrate
ET
TABLE 3 of Ferric Ammonium Citrate and Iodometric Method method
(mg)
Fe+++ found (mg)
Fe+++ (5)
50 40 30 20 10
lo.“5 1 8.21 6.16 4.10 2.05
20.50 20.53 20.53 20.50 20.50
a 1 ml 0.05 M sodium
thiosulfate
AL.
= 2.792
mg ferric
by
Iodometric
methoda
Fe+++ found
Fe+++
bd
(70)
10.47 5.38 6.34 4.19 2.07
20.94 20.95 21.13 20.95 20.70
iron.
Various known volumes ranging from 5 to 1 ml were treated in each case with 1 ml dilute sulfuric acid and were heated on an electric plate until the solution became pale yellow. The solution was then allowed to cool and an excess of 0.02 N n-ascorbic acid solution was added and the solution was shaken well. Then 5 ml of dilute sulfuric acid and 0.1 ml of methyl red indicator were added and 0.02 N N-bromosuccinimide solution was used for back-titration of excess standard L-ascorbic acid solution. A blank experiment was simultaneously carried out and the reading was subtracted from the titer before calculation. Simultaneously, comparative analysis of similar volumes was done by the iodometric met#hod using 0.05 N sodium thiosulfate. The results are recorded in Table 3. (B) Determination of ferric iron content of whole blood. An accurately measured volume of the blood sample, e.g., 1 ml, in a porcelain crucible of 20 ml capacity was ashed at 450°C for 7 hr in the muffle furnace. The ash was dissolved in 5 ml concentrated sulfuric acid and the solution was poured into 10 ml distilled water in a 100 ml stoppered Erlenmeyer flask. The crucible was washed twice with 5 ml distilled
Statistical
Analysis
of Results
TABLE 4 of Ferric Iron (mg per cent,)
Blood source”
ME&X.
Min.
Sheep Buff a10
53.60 50.25
37.97 33.50
(1 Each
group
included
sixty
animals.
Content,
of Whole
Blood
*SE. 46.55 43.55
0.56 0.57
FERRIC
Htat,istical
Analysis
IX
WHOLE
TABLE 5 of Blood Ferric (mg per cent)
of HesuIts
Blood source”
129
BLOOD
Iron
Content.
in Itelation
to Sex
Sex
Sheep
53.60 50.25 50.“5 -* 50,2F,
Xale Female Male Female
Buff alo
a Each
IRON
group
included
60 animals,
half from
39.08 37.97 35 74 33 50 males
4S.06 45 04 4.5.00 4”. 10
and half from
0. i6 0.71 0.54 0. 73
females.
water and the washings were transferred to the Erlenmeyer flask. A known volume of 0.01 AT L-ascorbic acid solution, e.g., 5 ml, was added and the mixture was shaken well for 2 min. The ascorbic acid solution must be in excess. Then 0.1 ml methyl red indicator was added and the excess L-ascorbic acid solution was back-titrated with 0.01 W N-bromosuccinimide solution. A blank experiment was simultaneously carried out and the reading subtracted from the titer before calculation. Statistical analysis of the results irrespective of age and sex was done to show the maximum, minimum, mean, and standard error. The results are given in Table 4. Also a statistical analysis of the result’s was made in relation to sex. The results are recorded in Table 5. Comparative analysis of the ferric iron content of 6 sheep blood samples from 6 individuals was simultaneously done on 1 ml of the
Comparat.ive
Analysis
TABLE 6 of Ferric Iron Content of Sheep Blood and Titanous Chloride Method Proposed
Sample No.~ 1 2 3 4 5 6
Fe+++/ml blood (mg) 0.5249 0.5137 0.5305 0.5249 0.5026 0.5305
method* Fe+++ (mg cT) 52.49 51.37 53.05 52.49 50.26 53.05
(1 One sample = one sheep. * 1 ml 0.01 N &ascorbic acid = 0.5584 mg ferric iron. c 1 ml 0.01 N tit,anous chloride = 0.5584 mg ferric iron.
by Proposed
Titanous Fe+++ /ml blood (mg) 0. 5137 0 5137 0.5249 0.5305 0 5026 0.5361
chloride
Method
method< Fe+++ (mg 51.37 51.37 52.49 53.05 50.26 53.61
%I
130
BARAKAT
ET
AL.
blood sample by the proposed method and the titanous chloride A 0.01 N n-ascorbic acid solution and 0.01 N titanous chloride were used, respectively. The results are shown in Table 6.
method. solution
DISCUSSION
Iodometric titration of ferric iron in solutions whose concentration is lower than 0.5 mg/ml shows no sharp end-point (5). The proposed method is a modification of a previous ascorbimetric method (3). The method is based on the fact that L-ascorbic acid readily reduces ferric iron in acid medium to ferrous iron, and is itself oxidized to dehydro-L-ascorbic acid. Thus a known volume of standard L-ascorbic acid solution in excess is added (A) and the excess ascorbic acid is back-titrated with standard N-bromosuccinimide solution (B) of the same normality (1) but using methyl red as an indicator. The end-point is easily detected when the red color of the indicator is just discharged (2). The volume of standard L-ascorbic acid solution equivalent to the ferric iron content of the unknown solution is easily calculated by difference (A - B). The proposed method is sensitive enough to determine quantities as low as 50 pg ferric iron (Table 1). The experimental error does not exceed k 2%. The method has been successfully applied for determining the ferric iron content of normal adult sheep and buffalo blood. Statistical analysis of the results has shown that normal adult sheep blood contains a maximum of 53.60 and a minimum of 37.97 with a mean ferric iron of 46.55 2 0.56 mg per cent. Normal adult buffalo blood has an average ferric iron value of 43.55 +0.57 with a maximum of 50.25 and a minimum of 33.50 mg per cent (Table 4). It is evident that female animals have a somewhat lower average value of ferric iron than males (Table 5). Comparative analysis of the ferric iron content of normal adult sheep blood has shown that the proposed method is analogous in accuracy to the recognized titanous chloride method. However, preparation of standard titanous chloride solution is tedious, since it requires special precautions and necessitates standardization before use (4). SUMMARY
A modified titrimetric method for the microdetermination of ferric iron with standard L-ascorbic acid is described. The mechanism of the reaction is discussed. The determination was carried out on quantities varying from 10 mg to 50 pg ferric iron. The experimental error does not exceed +-2%.
FERRIC
IRON
IN
WHOLE
BLOOD
131
A total of sixty samples of normal adult sheep and buffalo blood, half from males and half from females, were analyzed for the ferric iron content. Statistical analysis of the results shows the maximum, minimum, mean, and standard errors. Female animals show a lower average value of blood ferric iron content than males. Comparative analysis of the ferric iron content of sheep blood by the proposed method and the recognized titanous chloride method is reported. REFERENCES 1.
2. 3. 4.
5. 6.
M. Z. BARAKAT, M. F. ABD EL-WAHAB, AND M. M. EL-SADR, Anal. Chem. 27, 536 (1955). M. 2. BARAKAT AND M. SHAKER, An&& 89, 216 (1964). M. Z. BARIKAT, S. K. SHEHAB. AND N. AFIFI, Microchem. J. 8, 131 (1964). A. I. VOGEL, “A Text-book of Quantitative Inorganic Analysis,” 3rd ed., p. 329. Longmans, London, 1968. S. YAMAMOTO, Nippox Kngaku Zasshi 73, 668 (1952). C. YOSHIMURA AND T. FUJITANI, Nippon Kagnh Zasshi 76, 304 (1955).
47,
BIOCHEMISTRY
123-131
(1972)
Ascorbimetry: The Microdetermination
of Ferric
M. Z. BARAKAT, Biochemistry
Iron Content
M. BASSIOUNI, F. EL-EINANY
Department, Madina
Faculty Nasr,
Received
A. S. FAYZALLA,
of Medicine, Cairo, Egypt
August
of Whole
Azhar
Blood AND
University,
8, 1971
In 1964 a method was reported (3) for the microdetermination of ferric iron with standard L-ascorbic acid using ammonium thiocyanate indicator solution. Although the method is accurate and sensitive, its use is limited to the determination of ferric iron in pharmaceutical preparations. The present work describes a modification for determining the ferric iron content of whole blood. MATERIALS
AND
METHODS
A total of sixty samples of normal adult sheep and buffalo blood was analyzed for ferric iron content. Of these, thirty samples were withdrawn from normal males and thirty samples from normal females of each species. The age of the animals ranged from 2 to 5 years. Equipment
and Reagents
1. Microburet of 5 ml capacity graduated to 0.01 ml. 2. Graduated pipets of 1, 2, 5, and 10 ml capacity. 3. Volumetric flasks of 10 ml capacity. 4. Erlenmeyer flasks of 50 and 100 ml capacity. 5. Standard L-ascorbic acid, e.g., 0.02 X, freshly prepared aqueous solution. 6. Standard N-bromosuccinimide (NBS), freshly prepared aqueous solution, e.g., 0.02 N solution. 7. Dilute sulfuric acid, 10% (v/v). 8. Dilute hydrochloric acid, 10% (v/v). 9. Methyl red indicator solution, 0.04% (w/v), alcoholic solution. 10. Standard titanous chloride, e.g., 0.01 N solution. 123 @ 1972 by
Academic
Press,
Inc.
124 I’alidity
of the Keclctiou
for
Quantitntive
Determination
Eeforc the reaction hctwvcn bascorhic acid and ferric iron was applied for the dctcrminatioii of the ferric iron content of blood, it was verified quantitatively. An accurately mcasurcd volume, e.g., 5 ml of a solution containing 0.9644 gm (I mmolc) of ferric ammonium sulfate per 100 ml was placed in a 100 ml stoppercd I~~rlcnmcycr flask and treated with an equal volume of dilute hydrochloric acid. Then an excess known volume of an aqueous solution containing 0.176 gm (1 mmole) of L-ascorbic acid per 100 ml was added. The mist,ure was thoroughly shaken and 0.1 ml of methyl red indicator solution was added. The excess of standard L-ascorbic acid was dctormincd by hack-titration with a standard X-bromosuccinimide solution containing 0.178 gm (1 mmolc) per 100 ml. The standard :V-bromosuccinimide solution was added gradually from a microburet with continuous shaking until the red color of the indicator was just dischargecl. The volume of standard L-ascorbic acid solution equivalent to the volume of ferric ammonium sulfate solution was easily calculated hy subtracting the volume of the ,V-hromosuccinimide tit’er from the volume of L-ascorbic acid added. The reaction was found to he stoichiometric at room temperature and the results were as follows: Vol.
ferric
ammonimn
10 Vol.
L-ascorbic
acid
4
soln.
(1 mmolr/lOO
acid
10.05
soln.
added
(1 mmolc/lOO
ml)
3.97
takrn
4.03
I
(ml)
(A) :
used
(ml)
(ml)
(A - B) :
4
2.98 ml)
(ml): 2
6
(1 mmole/lOO
4.98
taken
3
8 soln.
ml)
ml)
5 (02
L-ascorbic
(1 mmole/lOO
5
N-bromosuccinimide 9.95
Vol.
soln.
10
20 Titer
sulfate
2 (B)
:
2.04
3.02
1.02
1.96
0.98
A similar series of experiments was done using a known volume of ferric ammonium sulfate solution containing double the number of molecules of solute (2 mmoles). An excess known volume of L-ascorbic acid solution (1 mmole) per 100 ml was added. It was found that the reaction was stoichiometric at room temperature. The results were: Vol.
ferric
ammonium
10 Vol.
L-ascorbic 30
acid
sulfatcl
soln.
(2 mmoles/lOO
5
4
soln.
(1 mmole/lOO
15
12
ml) 3
ml)
added 9
taken
(ml) 2
(ml)
: 1
(A) : 6
3
FERRIC
Titer
N-bromosuccinimide 9.Y8
IRON
WHOLE
soln. (1 mmole/lOO
4.YX
4
Vol. L-ascorbic acid soln. (1 mmole/100
20.02
Ih-
10.02
125
BLOOD
ml) used (ml)
2.0;2
3.0" ml) taken (ml)
0.96
(A - B) :
5.YH
8
(B) :
3.98
2.02
Procedure
To an accurately measured volume, e.g., 5 ml of an unknown aqueous ferric iron solution in a 100 ml stoppered Erlenmeyer flask, add an equal volume of dilute hydrochloric acid and a double volume of a standard L-ascorbic acid solution. The standard ascorbic acid solution must be in excess. Shake well the contents of the flask for a period of 2 min. Then add 0.1 ml of methyl red indicator and titrate back the excess of standard L-ascorbic acid solution with standard N-bromosuccinimide solution of the same normality added gradually from a microburet, with shaking after each addition until the red color is just discharged. Carry out a blank experiment simultaneously and subtract the reading from the titer before calculation. Calculate the ferric iron content of the unknown solution from the expression as follows : ferric iron content (mg or pg) = V X C X
55.84 x 2 176
1’ = volume of standard L-ascorbic acid solution taken in the reaction and C = concentration of L-ascorbic acid in mg or pg/ml solution. RESULTS
Microdetemination
Recovery of Ferric Iron
A stock aqueous solution containing 0.1 gm/lOO ml ferric iron was prepared by dissolving 0.8635 gm ferric ammonium sulfate in distilled water, and the volume was made up with distilled water to 100 ml in a volumetric flask. The ferric iron content was determined by the proposed method in various known volumes of the st,ock solution as if they were unknowns, using an excess of 0.02 N L-ascorbic acid solution for reduction of ferric ions and 0.02 N N-bromosuccinimide solution for back-titration of excess L-ascorbic acid. The ferric iron content of a IO-fold diluted solution was also determined in various known volumes as if they were unknowns. The results are shown in Table 1.
126
BARAKAT
Recovery
of Ferric
ET
AL.
TABLE 1 Iron by the Proposed
Method
o.oa Nn Ferric iron bd
0.02 N GAscorbic acid added (ml)
10 9 8 7 6 5 4 3 2 1
(A) 12 11 10 9 8 7 6 5 4 3
(ml) (A - R
Ferric iron found (me)
8.98 7.98 7.18 6.24 5.36 4.57 3.58 2.72 1.78 0.90
10.03 8.91 8.02 6.97 5.99 5.10 4.00 3.04 1.99 1.01
0.004 N (ml)
0.004 Nb (ml)
(Pd
5.44 4.95 4.40 3.90 3.32 2.80 2.20 1.66 1.10 0.56 0.78
4.56 4.05 3.60 3.10 2.68 2.20 1.80 1.34 0.90 0.44 0.22
0.02 N N-Bromosuccinimide used (ml) (B) 3.02 3.02 2.82 2.76 2.64 2.43 2.42 2.28 2.22 2.10 Dilution
(Pd
0.004 N (ml)
1000 900 800 700 600 500 400 300 200 100 50
10 9 8 7 6 5 4 3 2 1 1
a 1 ml 0.02 N Lascorbic b 1 ml 0.004 N n-ascorbic
Comparative
L-Ascorbic acid taken
Error (%) 0.30 1.00 0.25 0.43 0.17 2.00 1.33 0.50 1.00
1 X 10
acid = 1.1168 mg ferric acid = 223.36 pg ferric
1019 905 804 692 599 491 402 299 201 98 49
1.90 0.56 0.50 1.14 0.17 1.80 0.50 0.33 0.50 2.00 2.00
iron. iron.
Analysis of Ferric Iron by the Proposed and the Previ0.u Ascorbimetric Method
Method
The proposed method was compared with the previous ascorbimetric method (3) using ammonium thiocyanate as an indicator for determining quantities of ferric iron varying from 500 to 50 pg as if they were unknowns. A known volume of 0.004 N L-ascorbic acid solution was added in excess and 0.004 N N-bromosuccinimide solution was used for back-titration using methyl red indicator. The results are listed in Table 2.
FERRIC
Comparative
Fe+++ (/.G) 500 400 300 200 100
50
IRON
IN
WHOLE
127
BLOOD
TABLE 2 Analysis of Ferric Iron by Proposed Method Previous Ascorbimetric Method Fe+++ found by proposed method (&) 496 402 299 201 9s 49
Interfering
and
Error (%I
Fe+++ found by previous method (hcd
Error (%I
o.so 0.50 0.33 0.50 2.00 2.00
500 39s 302 204 101 49
0.50 0.67 2.00 1.00 2.00
Substances
Typical interfering ions include silver, mercuric, and cupric ions. The silver cations may be separated by dilute HCI in the form of insoluble silver chloride and removed by filtration. The mercuric ions may be converted into nonionizable soluble potassium mercuric iodide (K?HgI,) with an excess of potassium iodide. Ferric ions and cupric ions will liberate an equivalent amount of iodine. which can be titrated with standard L-ascorbic acid solution using starch as an indicator (A). Sodium fluoride can be used to mask ferric ions in the presence of a few drops of ammonium thiocyanate solution until the red color disappears (6). Cupric ions wit,11 excess potassium iodide form cupric iodide, which decomposes spontaneously to cuprous iodide and free iodine, which can be titrated with the same standard L-ascorbic acid using starch indicator (B). The difference between the two titers (A B) corresponds to the ferric iron in the volume taken. Experimental
Error
The experimental error of the proposed method does not exceed ,270 when determining amounts varying from 10 mg to 50 /kg of ferric iron (Tables 1 and 2). Application
of the Proposed Method
(A) Determination of fewic iron content of ferric ammonium citrate. An accurately weighed amount of ferric ammonium citrate (in form of scales), e.g., 1 gm was brought into aqueous solution and the volume was completed with distilled water to 100 ml in a volumetric flask, so that 1 ml of the solution was equivalent to 10 mg ferric ammonium citrate.
128
BARAKAT
Recovery
of Ferric Iron Proposed Method Proposed
Ferric ammonium citrate
ET
TABLE 3 of Ferric Ammonium Citrate and Iodometric Method method
(mg)
Fe+++ found (mg)
Fe+++ (5)
50 40 30 20 10
lo.“5 1 8.21 6.16 4.10 2.05
20.50 20.53 20.53 20.50 20.50
a 1 ml 0.05 M sodium
thiosulfate
AL.
= 2.792
mg ferric
by
Iodometric
methoda
Fe+++ found
Fe+++
bd
(70)
10.47 5.38 6.34 4.19 2.07
20.94 20.95 21.13 20.95 20.70
iron.
Various known volumes ranging from 5 to 1 ml were treated in each case with 1 ml dilute sulfuric acid and were heated on an electric plate until the solution became pale yellow. The solution was then allowed to cool and an excess of 0.02 N n-ascorbic acid solution was added and the solution was shaken well. Then 5 ml of dilute sulfuric acid and 0.1 ml of methyl red indicator were added and 0.02 N N-bromosuccinimide solution was used for back-titration of excess standard L-ascorbic acid solution. A blank experiment was simultaneously carried out and the reading was subtracted from the titer before calculation. Simultaneously, comparative analysis of similar volumes was done by the iodometric met#hod using 0.05 N sodium thiosulfate. The results are recorded in Table 3. (B) Determination of ferric iron content of whole blood. An accurately measured volume of the blood sample, e.g., 1 ml, in a porcelain crucible of 20 ml capacity was ashed at 450°C for 7 hr in the muffle furnace. The ash was dissolved in 5 ml concentrated sulfuric acid and the solution was poured into 10 ml distilled water in a 100 ml stoppered Erlenmeyer flask. The crucible was washed twice with 5 ml distilled
Statistical
Analysis
of Results
TABLE 4 of Ferric Iron (mg per cent,)
Blood source”
ME&X.
Min.
Sheep Buff a10
53.60 50.25
37.97 33.50
(1 Each
group
included
sixty
animals.
Content,
of Whole
Blood
*SE. 46.55 43.55
0.56 0.57
FERRIC
Htat,istical
Analysis
IX
WHOLE
TABLE 5 of Blood Ferric (mg per cent)
of HesuIts
Blood source”
129
BLOOD
Iron
Content.
in Itelation
to Sex
Sex
Sheep
53.60 50.25 50.“5 -* 50,2F,
Xale Female Male Female
Buff alo
a Each
IRON
group
included
60 animals,
half from
39.08 37.97 35 74 33 50 males
4S.06 45 04 4.5.00 4”. 10
and half from
0. i6 0.71 0.54 0. 73
females.
water and the washings were transferred to the Erlenmeyer flask. A known volume of 0.01 AT L-ascorbic acid solution, e.g., 5 ml, was added and the mixture was shaken well for 2 min. The ascorbic acid solution must be in excess. Then 0.1 ml methyl red indicator was added and the excess L-ascorbic acid solution was back-titrated with 0.01 W N-bromosuccinimide solution. A blank experiment was simultaneously carried out and the reading subtracted from the titer before calculation. Statistical analysis of the results irrespective of age and sex was done to show the maximum, minimum, mean, and standard error. The results are given in Table 4. Also a statistical analysis of the result’s was made in relation to sex. The results are recorded in Table 5. Comparative analysis of the ferric iron content of 6 sheep blood samples from 6 individuals was simultaneously done on 1 ml of the
Comparat.ive
Analysis
TABLE 6 of Ferric Iron Content of Sheep Blood and Titanous Chloride Method Proposed
Sample No.~ 1 2 3 4 5 6
Fe+++/ml blood (mg) 0.5249 0.5137 0.5305 0.5249 0.5026 0.5305
method* Fe+++ (mg cT) 52.49 51.37 53.05 52.49 50.26 53.05
(1 One sample = one sheep. * 1 ml 0.01 N &ascorbic acid = 0.5584 mg ferric iron. c 1 ml 0.01 N tit,anous chloride = 0.5584 mg ferric iron.
by Proposed
Titanous Fe+++ /ml blood (mg) 0. 5137 0 5137 0.5249 0.5305 0 5026 0.5361
chloride
Method
method< Fe+++ (mg 51.37 51.37 52.49 53.05 50.26 53.61
%I
130
BARAKAT
ET
AL.
blood sample by the proposed method and the titanous chloride A 0.01 N n-ascorbic acid solution and 0.01 N titanous chloride were used, respectively. The results are shown in Table 6.
method. solution
DISCUSSION
Iodometric titration of ferric iron in solutions whose concentration is lower than 0.5 mg/ml shows no sharp end-point (5). The proposed method is a modification of a previous ascorbimetric method (3). The method is based on the fact that L-ascorbic acid readily reduces ferric iron in acid medium to ferrous iron, and is itself oxidized to dehydro-L-ascorbic acid. Thus a known volume of standard L-ascorbic acid solution in excess is added (A) and the excess ascorbic acid is back-titrated with standard N-bromosuccinimide solution (B) of the same normality (1) but using methyl red as an indicator. The end-point is easily detected when the red color of the indicator is just discharged (2). The volume of standard L-ascorbic acid solution equivalent to the ferric iron content of the unknown solution is easily calculated by difference (A - B). The proposed method is sensitive enough to determine quantities as low as 50 pg ferric iron (Table 1). The experimental error does not exceed k 2%. The method has been successfully applied for determining the ferric iron content of normal adult sheep and buffalo blood. Statistical analysis of the results has shown that normal adult sheep blood contains a maximum of 53.60 and a minimum of 37.97 with a mean ferric iron of 46.55 2 0.56 mg per cent. Normal adult buffalo blood has an average ferric iron value of 43.55 +0.57 with a maximum of 50.25 and a minimum of 33.50 mg per cent (Table 4). It is evident that female animals have a somewhat lower average value of ferric iron than males (Table 5). Comparative analysis of the ferric iron content of normal adult sheep blood has shown that the proposed method is analogous in accuracy to the recognized titanous chloride method. However, preparation of standard titanous chloride solution is tedious, since it requires special precautions and necessitates standardization before use (4). SUMMARY
A modified titrimetric method for the microdetermination of ferric iron with standard L-ascorbic acid is described. The mechanism of the reaction is discussed. The determination was carried out on quantities varying from 10 mg to 50 pg ferric iron. The experimental error does not exceed +-2%.
FERRIC
IRON
IN
WHOLE
BLOOD
131
A total of sixty samples of normal adult sheep and buffalo blood, half from males and half from females, were analyzed for the ferric iron content. Statistical analysis of the results shows the maximum, minimum, mean, and standard errors. Female animals show a lower average value of blood ferric iron content than males. Comparative analysis of the ferric iron content of sheep blood by the proposed method and the recognized titanous chloride method is reported. REFERENCES 1.
2. 3. 4.
5. 6.
M. Z. BARAKAT, M. F. ABD EL-WAHAB, AND M. M. EL-SADR, Anal. Chem. 27, 536 (1955). M. 2. BARAKAT AND M. SHAKER, An&& 89, 216 (1964). M. Z. BARIKAT, S. K. SHEHAB. AND N. AFIFI, Microchem. J. 8, 131 (1964). A. I. VOGEL, “A Text-book of Quantitative Inorganic Analysis,” 3rd ed., p. 329. Longmans, London, 1968. S. YAMAMOTO, Nippox Kngaku Zasshi 73, 668 (1952). C. YOSHIMURA AND T. FUJITANI, Nippon Kagnh Zasshi 76, 304 (1955).