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Spectrophotometric determination of iron with orthophenanthroline
MICROCHEMICAL
JOURNAL
15,
585-589
(1970)
Spectrophotometric Determination with Orthophenanthroline G. S. R. KRISHNA Dil.isicm
MURTI,
of Agricult~cml
A. V. MOHARIR,
of Iron
AND
Piz~.~ics, I~~tliatr Agriculrrtr-al Nell% Dcllri, I&in
V. A. K. SARMA Rrsenrci~
Institute,
Orthophenanthroline forms with ferrous iron in aqueous solution a soluble orange-red complex, stable between pH 2 and 9 (IO). Measurement of the color intensity at 5 1S-540 m/l, has been widely used for the determination of traces of iron in reagent chemicals (I I ), biological materials (S), plant extracts (/, .?). and citric acid extracts from soils (7). Several reagents have been suggested for the reduction of ferric iron prior to formation of the complex. Of these, hydroxylamine hydrochloride has been considered to bc the most efficient (2). However, for systems containing citrate, hydroxylamine hydrochloride has been variously reported to bc unsuitable (I) and quite suitable (7). The accuracy of the calorimetric determination of iron with orthophenanthroline depends on (a) completeness of reduction of Fe”+ to Fe’-: (b) pH of iron solution bcforc complex formation, (c) stability of the colored complex, and (d) interference of other ions in the formation of the color. Several reagents have been tried as reductants. Sodium dithionite, though reducing iron completely. introduces turbidity in the test solution within a few minutes (5). Sodium sulfite and formaldehyde complex with Fe’: and thus cause considerable error in the determination (2). Sodium and potassium formates lead to error due to formic acid complexing with Fc:l+ (13). The ferrous iron-orthophenanthrolinc complex, once formed, is stable even up to 15 days. and has constant color intensity between pH 2 and 9 (IO, 9). It has been found that the intensity of the color increases with time if the pH of the iron solution is adjusted to a value >4 before reduction at room temperature with hydroxylamine hydrochloride, indicating incompleteness of reduction ( 7). Complete reduction of the iron can, however, be achieved in such solutions by heating the solution with the reductant and a pH 3.5 acetate buffer for 1 hour at 65°C
(7).
It has been reported that most anions and cations, up to a concentra585
586
KRISHNA
MURTI,
MOHARIR,
AND
SARMA
tion 250 times that of iron, do not interfere with iron determination by orthophenanthroline (2). No common soil ions except orthophosphate are considered to interfere in the determination (6). The methods recommended by earlier workers are time consuming and difficult to apply when the number of samples is large. It was, therefore, considered necessary to develop a rapid and reliable modification of the method. Thioglycolic acid has been successfully used to reduce iron to eliminate interference in the calorimetric determination of zirconium (4). It was thought worthwhile to investigate the suitability of thioglycolic acid as reductant in the spectrophotometric determination of iron with orthophenanthroline. The procedure so developed is presented below. MATERIALS
AND
METHODS
Iron standards were obtained by dilution of 100 ppm iron stock solution prepared from spectroscopically pure ferric oxide. A Lumetron Model 402-E photoelectric calorimeter with a narrow band 515 rnp monochromatic filter was used for measurement of transmittance of the solutions after development of color. Proposed method. A suitable aliquot (< 150 ,Lg of Fe) of the solution is taken in a 50-ml volumetric flask and 10 ml of (pH 3.5) M sodium citrate-citric acid buffer, followed by 1 ml of 4% thioglycolic acid, is added. The contents of the flask are mixed well, 2 ml of 0.4% orthophenanthroline are added, and the volume is made up to 50 ml. Transmittance of the solution is measured at 515 rnp after 5 minutes. A standard curve in the range O-150 pg of Fe is prepared in the same way using aliquots of standard iron solution, and the iron content of the test solution is read from the curve. RESULTS
AND
DISCUSSION
The optical densities obtained with different amounts of iron are presented in Table 1 and show that Beer’s law is obeyed in the range O-3 ppm Fe. Development of color was instantaneous, and the color of the complex was stable for 72 hours. EfJect of pH Aliquots of standard iron solution representing 62.5 pg of Fe were buffered with 15 ml of citrate-citric acid buffers of different pH values, and the amount of iron was determined by the proposed method. Table 2 gives the amount of iron recovered from each solution and the final pH of the solution. Complete recovery in all cases showed that reduction by thioglycolic acid was complete at all the pH values studied. In
DETERMINATION
OF
TAfSf.fOPWAL
Dmstrtts
OF %AVDAIW I ttt.
I
ftwN
Pruwost
II
I-inal
5x7
IRON
sOt.UTIONS
,zr 515 tnP Umt;
MLTIIOD
iroti
C‘OiIC No.
( ,‘,,I11 1
I
0.25
7
0.50
3
I .oo
1
I
5
2.00
io
6
2.50
1
i .oo
the proposed method, however, addition of pH 3.5 buffer is rccommended to prevent the possible precipitation of phosphates from test solutions. Aliquots of standard iron solution representing 50 /kg of Fc were adjusted to different pH values between 2 and 9 by predetermined aliquots of 0.1 N NaOH and 0.1 N HCI. The iron in the solutions was then determined by the present method and the methods of Olson (9) and Krishna Murti et al. (7). The results, given in Table 3, show that complete recovery of iron was obtained using the method of Krishna Murti et al. (7) and the present method. Olson’s (9) method gave extremely low values for iron when the pH of the solution was greater than 2. In a solution of pH higher than 4, the iron forms hydroxylated complexes which are difficult to reduce (7). Hydroxylamine hydrochloride is capable of reducing the iron only with heating at 65°C for 1 hour (7). Thioglycolic acid, however, reduced the iron instantly at room temperature at any pH.
588
KRISHNA
MURTI,
MOHARIR,
AND
SARMA
Interference of Other Ions
Suitable aliquots representing various concentrations of several other ions in the final solution were taken with 50 /.lg of iron and the amount of iron was determined by the proposed method. No interference of Ca (10,000 ppm), Mg (1200 ppm), Al (150 ppm), Ti (80 ppm), Zr (50 ppm>, PO4 (30 ppm), Mn (20 ppm), Ba (20 ppm), Zn (20 ppm), Cu (2 ppm), and Ni (0.6 ppm) was found in the determination of iron with the proposed method. Free Iron Oxides Extracted from Soil Clay
The free iron oxide content in the clay fraction (<2 p of two soils from Madhya Pradesh, India, were extracted by the dithionite-citrate-bicarbonate method of Mehra and Jackson (8). Suitable aliquots of the extract were taken and the iron was determined by the present method and the method of Krishna Murti et al. (7). The amounts of iron obtained are presented in Table 4. The closenessof the values show that the proposed method is reliable and accurate for such determinations. TABLE
EFFECT OF INITIAL pH
3
OF IRON SOLUTION ON THE DETERMINATION OF IRON BY VARIOUS METHODS
Amount of iron found (,~g) Initial pH of iron
No.
Amount of iron taken h!
solution a
(1965)
Krishna Murti et al. (1966)
Proposed method
1 2 3 4
50.0 50.0 50.0 50.0
2.0 4.0 6.0 9.0
50.0 20.5 15.0 0
50.0 49.5 50.0 49.5
50.0 50.0 50.0 50.0
Olson
a pH adjusted by 0.1 N HCl or 0.1 N NaOH
TABLE 4 DETERMINATION OF IRON IN EXTRACIX OF FREE IRON OXIDES FROM CLAY FRACTIONS OF Sons
Amount of iron found (pg) No.
Clay sample
Proposed method
Krishna Murti ef al.
1 2
Umaria Raogarh
97.5 64.5
96.5 64.5
DETERMINATION
OF
IRON
589
SUMMARY A modified procedure is presented for the spectrophotometric determination of iron by orthophenanthroline. Reduction of iron prior to development of color is accomplished by thioglycolic acid, and is instantaneous at room temperature. The system is buffered at pH 3.5 with sodium citrate-citric acid to avoid precipitation of hydroxides and phosphates from the test solution. Moderate amounts of Ca, Mg, Al, Ti, Zr. PO,, Mn, Ba. Zn are permissible but more than 2 ppm of Cu and 0.6 ppm of Ni interfere. REFERENCES 1. Cowling, H.. and Benne, E. J.. Report on zinc and iron in plants. J. Ass. Ofl. A,w. Citrrn. 25, 55.5-567 (1942).
2. Fortune, W. B., and Mellon. M. G.. Determination of iron with o-phenanthroline. 111(i.E/l,?. Cher17.. Ann/. Ed. 10, 60-64 (1938). 3. Gupta? U. C., Studies on the o-phenanthroline method for determination of iron in plant materials. P/c//rt Soil 28, 298-305 (1968). 4. Hahn. R. B.. Zirconium and hafnium. 112“Treatise on Analytical Chemistry” (I. M. Kolthoff, P. J. Elving with E. B. Sandell, eds.), Part II, Vol. 5, pp. 61-13X. Wiley (Interscience). New York, 1961. 5. Hummel, F. C.. and Willard. H. H.. Determination of iron in biological materials. The use of o-phenanthroline. It&. Et/g. Chrnr., And. Etf. 10, 13-15 (1938). 6. Jackson. M. L.. “Soil Chemical Analysi\,” 498 pp. Prentice-Hall, Englewood Cliffs, N. J., 1958. 7. Krishna Murti. G. S. R.. Volk. V. V.. and Jackson, M. L. Colorimetrjc determination of iron of mixed valency by orthophenanthroline. Soil Sci. SM. Amer., P,oc. 29, 663-663
(1966).
8. hlehra. 0. P.. and Jackson. M. L., Iron oxide removal from soils and clays by a dithionite-citrate system buffered with sodium bicarbonate. Clays Cluy Mirfc~r., Proc,. Naf. Co~r/. 7th 1960, 3 17-327.
9. Olson, R. V.. Iron. Irz “Methods of Soil Analysis“ (C. A. Black, ed.), Part 2. pp. 963-973. Amer. Sot. Agronomy, Madison, Wis., 1965. 10. Sandell, E. B., “Calorimetric Determination of Traces of Metals.” 487 pp. Wiley (Interscience). New York, 1944. II. Stoke\. H. N.. and Cain. I. R., Determination of traces of iron in reagent chemicals and its isolation. .l. ~lmc~.. C/fern. Sot. 29, 409-443 (1907).
JOURNAL
15,
585-589
(1970)
Spectrophotometric Determination with Orthophenanthroline G. S. R. KRISHNA Dil.isicm
MURTI,
of Agricult~cml
A. V. MOHARIR,
of Iron
AND
Piz~.~ics, I~~tliatr Agriculrrtr-al Nell% Dcllri, I&in
V. A. K. SARMA Rrsenrci~
Institute,
Orthophenanthroline forms with ferrous iron in aqueous solution a soluble orange-red complex, stable between pH 2 and 9 (IO). Measurement of the color intensity at 5 1S-540 m/l, has been widely used for the determination of traces of iron in reagent chemicals (I I ), biological materials (S), plant extracts (/, .?). and citric acid extracts from soils (7). Several reagents have been suggested for the reduction of ferric iron prior to formation of the complex. Of these, hydroxylamine hydrochloride has been considered to bc the most efficient (2). However, for systems containing citrate, hydroxylamine hydrochloride has been variously reported to bc unsuitable (I) and quite suitable (7). The accuracy of the calorimetric determination of iron with orthophenanthroline depends on (a) completeness of reduction of Fe”+ to Fe’-: (b) pH of iron solution bcforc complex formation, (c) stability of the colored complex, and (d) interference of other ions in the formation of the color. Several reagents have been tried as reductants. Sodium dithionite, though reducing iron completely. introduces turbidity in the test solution within a few minutes (5). Sodium sulfite and formaldehyde complex with Fe’: and thus cause considerable error in the determination (2). Sodium and potassium formates lead to error due to formic acid complexing with Fc:l+ (13). The ferrous iron-orthophenanthrolinc complex, once formed, is stable even up to 15 days. and has constant color intensity between pH 2 and 9 (IO, 9). It has been found that the intensity of the color increases with time if the pH of the iron solution is adjusted to a value >4 before reduction at room temperature with hydroxylamine hydrochloride, indicating incompleteness of reduction ( 7). Complete reduction of the iron can, however, be achieved in such solutions by heating the solution with the reductant and a pH 3.5 acetate buffer for 1 hour at 65°C
(7).
It has been reported that most anions and cations, up to a concentra585
586
KRISHNA
MURTI,
MOHARIR,
AND
SARMA
tion 250 times that of iron, do not interfere with iron determination by orthophenanthroline (2). No common soil ions except orthophosphate are considered to interfere in the determination (6). The methods recommended by earlier workers are time consuming and difficult to apply when the number of samples is large. It was, therefore, considered necessary to develop a rapid and reliable modification of the method. Thioglycolic acid has been successfully used to reduce iron to eliminate interference in the calorimetric determination of zirconium (4). It was thought worthwhile to investigate the suitability of thioglycolic acid as reductant in the spectrophotometric determination of iron with orthophenanthroline. The procedure so developed is presented below. MATERIALS
AND
METHODS
Iron standards were obtained by dilution of 100 ppm iron stock solution prepared from spectroscopically pure ferric oxide. A Lumetron Model 402-E photoelectric calorimeter with a narrow band 515 rnp monochromatic filter was used for measurement of transmittance of the solutions after development of color. Proposed method. A suitable aliquot (< 150 ,Lg of Fe) of the solution is taken in a 50-ml volumetric flask and 10 ml of (pH 3.5) M sodium citrate-citric acid buffer, followed by 1 ml of 4% thioglycolic acid, is added. The contents of the flask are mixed well, 2 ml of 0.4% orthophenanthroline are added, and the volume is made up to 50 ml. Transmittance of the solution is measured at 515 rnp after 5 minutes. A standard curve in the range O-150 pg of Fe is prepared in the same way using aliquots of standard iron solution, and the iron content of the test solution is read from the curve. RESULTS
AND
DISCUSSION
The optical densities obtained with different amounts of iron are presented in Table 1 and show that Beer’s law is obeyed in the range O-3 ppm Fe. Development of color was instantaneous, and the color of the complex was stable for 72 hours. EfJect of pH Aliquots of standard iron solution representing 62.5 pg of Fe were buffered with 15 ml of citrate-citric acid buffers of different pH values, and the amount of iron was determined by the proposed method. Table 2 gives the amount of iron recovered from each solution and the final pH of the solution. Complete recovery in all cases showed that reduction by thioglycolic acid was complete at all the pH values studied. In
DETERMINATION
OF
TAfSf.fOPWAL
Dmstrtts
OF %AVDAIW I ttt.
I
ftwN
Pruwost
II
I-inal
5x7
IRON
sOt.UTIONS
,zr 515 tnP Umt;
MLTIIOD
iroti
C‘OiIC No.
( ,‘,,I11 1
I
0.25
7
0.50
3
I .oo
1
I
5
2.00
io
6
2.50
1
i .oo
the proposed method, however, addition of pH 3.5 buffer is rccommended to prevent the possible precipitation of phosphates from test solutions. Aliquots of standard iron solution representing 50 /kg of Fc were adjusted to different pH values between 2 and 9 by predetermined aliquots of 0.1 N NaOH and 0.1 N HCI. The iron in the solutions was then determined by the present method and the methods of Olson (9) and Krishna Murti et al. (7). The results, given in Table 3, show that complete recovery of iron was obtained using the method of Krishna Murti et al. (7) and the present method. Olson’s (9) method gave extremely low values for iron when the pH of the solution was greater than 2. In a solution of pH higher than 4, the iron forms hydroxylated complexes which are difficult to reduce (7). Hydroxylamine hydrochloride is capable of reducing the iron only with heating at 65°C for 1 hour (7). Thioglycolic acid, however, reduced the iron instantly at room temperature at any pH.
588
KRISHNA
MURTI,
MOHARIR,
AND
SARMA
Interference of Other Ions
Suitable aliquots representing various concentrations of several other ions in the final solution were taken with 50 /.lg of iron and the amount of iron was determined by the proposed method. No interference of Ca (10,000 ppm), Mg (1200 ppm), Al (150 ppm), Ti (80 ppm), Zr (50 ppm>, PO4 (30 ppm), Mn (20 ppm), Ba (20 ppm), Zn (20 ppm), Cu (2 ppm), and Ni (0.6 ppm) was found in the determination of iron with the proposed method. Free Iron Oxides Extracted from Soil Clay
The free iron oxide content in the clay fraction (<2 p of two soils from Madhya Pradesh, India, were extracted by the dithionite-citrate-bicarbonate method of Mehra and Jackson (8). Suitable aliquots of the extract were taken and the iron was determined by the present method and the method of Krishna Murti et al. (7). The amounts of iron obtained are presented in Table 4. The closenessof the values show that the proposed method is reliable and accurate for such determinations. TABLE
EFFECT OF INITIAL pH
3
OF IRON SOLUTION ON THE DETERMINATION OF IRON BY VARIOUS METHODS
Amount of iron found (,~g) Initial pH of iron
No.
Amount of iron taken h!
solution a
(1965)
Krishna Murti et al. (1966)
Proposed method
1 2 3 4
50.0 50.0 50.0 50.0
2.0 4.0 6.0 9.0
50.0 20.5 15.0 0
50.0 49.5 50.0 49.5
50.0 50.0 50.0 50.0
Olson
a pH adjusted by 0.1 N HCl or 0.1 N NaOH
TABLE 4 DETERMINATION OF IRON IN EXTRACIX OF FREE IRON OXIDES FROM CLAY FRACTIONS OF Sons
Amount of iron found (pg) No.
Clay sample
Proposed method
Krishna Murti ef al.
1 2
Umaria Raogarh
97.5 64.5
96.5 64.5
DETERMINATION
OF
IRON
589
SUMMARY A modified procedure is presented for the spectrophotometric determination of iron by orthophenanthroline. Reduction of iron prior to development of color is accomplished by thioglycolic acid, and is instantaneous at room temperature. The system is buffered at pH 3.5 with sodium citrate-citric acid to avoid precipitation of hydroxides and phosphates from the test solution. Moderate amounts of Ca, Mg, Al, Ti, Zr. PO,, Mn, Ba. Zn are permissible but more than 2 ppm of Cu and 0.6 ppm of Ni interfere. REFERENCES 1. Cowling, H.. and Benne, E. J.. Report on zinc and iron in plants. J. Ass. Ofl. A,w. Citrrn. 25, 55.5-567 (1942).
2. Fortune, W. B., and Mellon. M. G.. Determination of iron with o-phenanthroline. 111(i.E/l,?. Cher17.. Ann/. Ed. 10, 60-64 (1938). 3. Gupta? U. C., Studies on the o-phenanthroline method for determination of iron in plant materials. P/c//rt Soil 28, 298-305 (1968). 4. Hahn. R. B.. Zirconium and hafnium. 112“Treatise on Analytical Chemistry” (I. M. Kolthoff, P. J. Elving with E. B. Sandell, eds.), Part II, Vol. 5, pp. 61-13X. Wiley (Interscience). New York, 1961. 5. Hummel, F. C.. and Willard. H. H.. Determination of iron in biological materials. The use of o-phenanthroline. It&. Et/g. Chrnr., And. Etf. 10, 13-15 (1938). 6. Jackson. M. L.. “Soil Chemical Analysi\,” 498 pp. Prentice-Hall, Englewood Cliffs, N. J., 1958. 7. Krishna Murti. G. S. R.. Volk. V. V.. and Jackson, M. L. Colorimetrjc determination of iron of mixed valency by orthophenanthroline. Soil Sci. SM. Amer., P,oc. 29, 663-663
(1966).
8. hlehra. 0. P.. and Jackson. M. L., Iron oxide removal from soils and clays by a dithionite-citrate system buffered with sodium bicarbonate. Clays Cluy Mirfc~r., Proc,. Naf. Co~r/. 7th 1960, 3 17-327.
9. Olson, R. V.. Iron. Irz “Methods of Soil Analysis“ (C. A. Black, ed.), Part 2. pp. 963-973. Amer. Sot. Agronomy, Madison, Wis., 1965. 10. Sandell, E. B., “Calorimetric Determination of Traces of Metals.” 487 pp. Wiley (Interscience). New York, 1944. II. Stoke\. H. N.. and Cain. I. R., Determination of traces of iron in reagent chemicals and its isolation. .l. ~lmc~.. C/fern. Sot. 29, 409-443 (1907).