The microdetermination of ferrous iron

MICROCHEMICAL

JOURNAL

The

(1964)

Microdetermination

A~OHAMED Biochemistry

8, 6-11

ZAKI

BARAKAT

Department, Faculty G&a, Cairo, Received

of AND

SAAD

of Vetekavy United Arab

November

Ferrous KHALIL

Medicine, Republic

Iron SHEHAB Cairo

University,

29, 1963

IKTRODUCTION

Several published methods for the determination of ferrous iron yield unsatisfactory results. In determining ferrous iron by titration with potassium dichromate, both potassium ferricyanide as external indicator and diphenylamine as internal indicator yield high results in the presence of carbohydrates (2). The method described in the 1936 addendum to the 1932 British Pharmacopoeia for the assay of ferrous iron is shown to give results about 10% too high (I). Chloramine B as a volumetric reagent loses 2-3%) of its oxidizing ability every 2 weeks (5) : as a bromometric reagent the end point is not sharp (3). The permanganate method (British Pharmacopoeia 1958) or the ceric sulfate method (United States Pharmacopoeia XVI and NF XI) are both titrimetric methods which recommend the determination of about 1 g of ferrous sulfate; otherwise the error increases with dilution of the ferrous solution. The present work describes a photoelectric calorimetric method for the determination of quantities as low as 20 ug of ferrous iron. MATERIALS

Equipment

.4SD

METHODS

and Reagents

1. EEL absorptiometer (Evans Electroselenium Ltd., Harlow Essex, England). 2. Graduated pipettes of l-, 2, 5-, and IO-ml capacity. 3. Volumetric flasks of 100ml-capacity. 4. Ammonium molybdate reagent prepared by dissolving 5 g of ammonium molybdate in 80 ml of distilled water and then adding

MICRODETERMINATION

OF

FERROUS

IRON

7

20 ml of orthophosphoric acid. This reagent is freshly prepared and kept in a brown bottle. 5. Standard stock solution of ferrous iron prepared by dissolving 0.2482 g of crystalline ferrous sulfate (Fe SO, * 7 H20) (equivalent to 50 mg of ferrous iron) in distilled water, then adding 10 ml of dilute sulfuric acid (lo%,, v,‘v), and completing the volume with distilled water to 100 ml in a volumetric flask. Procedure Into a loo-ml volumetric flask is added X ml of the standard stock solution of ferrous iron (1 ml represents 500 ltg of ferrous iron). Then 10 ml of dilute sulfuric acid (lo?, v/v) are added and the volume is diluted to 100 ml with distilled water, mixed well, and left to stand for about 5 minutes. Five ml of this solution is then transferred to a dry calorimeter cup and 5 ml of the ammonium molybdate reagent added. The mixture is mixed well and its percentage transmission read after 5 minutes in the EEL absorptiometer by using a green filter 604 (peak response being 520 mlt) with a mixture of 5 ml of the reagent and 5 ml of distilled water, set at 1007; transmission. S = 20 ml of the standard stock solution of ferrous iron (50 mg/lOO ml) representing a concentration of 500 ng of ferrous iron per 5 ml of solution or 16, 12: 8, and 4 ml of the standard stock solution of ferrous iron representing concentrations of 400, 300, 200, and 100 ltg of ferrous iron per 5 ml of solution. To obtain lower concentrations, 2, 1.6, 1.2, 0.8, and 0.4 ml of the standard stock ferrous iron solution were diluted with distilled water to 100 ml in a standard flask, and represented concentrations of 50, 40, 30. 20, and 10 ILg of ferrous iron per 5 ml of solution. From the results obtained it was found that (a) transmission readings should be spread out sufficiently to allow a determination to be made within concentrations ranging from 20 to 500 ug of ferrous iron per 5 ml, and (b) since the graph shows that within these concentrations there is a slight deviation from Beer’s Law, a calibration table can replace the graph and give more accurate results, provided that the estimations are carried out at the same (room) temperature and under the same conditions. Calibration

Table

From the standard stock solution of ferrous iron (50 mg/lOO ml) standard solutions are prepared so that 5 ml of each dilution contains an amount of ferrous iron ranging from 100 to 500 LIP> increasing in the order of

8

M.

2. BARAKAT

AND

S. I<. SHEHAB

100 ug; 5 ml of each dilution is accurately measuredinto a dry calorimeter cup; 5 ml of ammonium molybdate reagent is added, mixed well, and left to stand for about 5 minutes. The percentage transmissionof the solution is then read. The results obtained at room temperature (203C) are shown in Table 1. TABLE CALIBRATION

Ferrous (w)

iron

Transmission (%)

500 400 300 200 100

1 TABLE

Ferrous iron (I%)

Transmission (%I

12

so

85

19 29 44 67

40 30 20 10

88 92 9s 98

Since somescientific investigators prefer to use absorbance rather than percentage transmission in standard curves or calibration tables, the absorbancevalue is plotted against concentration (Fig. 1) , illustrating the

1.0 0.9 0.6 0.7 0.6 I-: 0.5 y 0.4 0.3 0.2 0. I 0 Concentration FIG. 1.

Standard

curve

showing

the relation

of Fe” .in microgmms between

concentration

and absorbancy.

best straight line when using various concentrations ranging from 100 to 500 ug of ferrous iron per 5 ml.

MICRODETERMINATION

OF

FERROUS

1RON

9

Method o j Assay Test solutions: ,4 dilution of the test solution is made so that 5 ml contains between 20 and 500 pg of ferrous iron. Then 5 ml of this solution is accurately measured into a dry calorimeter cup, 5 ml of ammonium molybdate reagent is added, mixed, and left to stand for 5 minutes. The percentage transmission of the solution is measured and the amount of ferrous iron read from the calibration table. Syrups: A known volume or weight of the syrup is diluted with distilled water in a volumetric flask so that 5 ml contains between 100 and 500 ltg of ferrous iron. Five ml of this dilution is accurately measured and introduced into a dry calorimeter cup: 5 ml of ammonium molybdate reagent is added and mixed well. The percentage transmission of the solution is measured and the concentration read from the calibration table. The figure obtained is the amount in micrograms contained in 5 ml of the diluted solution. The deviation does not exceed 10%. Tablets: Ten tablets are accurately weighed and the average weight of a single tablet determined. Then the tablets are finely powdered in a dry mortar and mixed well. An amount corresponding to one tablet is accurately weighed and digested with 20 ml of a 1476 (v/v) solution of sulfuric acid until no more than a small residue remains. The solution is filtered into a loo-ml volumetric flask and the residue is washed with a sufficient amount of the diluted sulfuric acid (about 10 ml) ; the volume is brought to the mark with distilled water. Suitable dilutions are then made according to the ferrous iron content in the tablet. The deviation is not more than 15%. RESULTS

Interfering

Substances

Carbohydrates do not interfere with the reaction, and in particular with such reducing sugars as glucose, fructose, galactose, lactose, and maltose. Furthermore, no interference could be observed with most watersoluble vitamins such as thiamin chloride, riboflavine, pyridoxine, nicotinic acid, and nicotinamide. The only interfering substance which develops a faint blue color with the reagent on standing for 10 minutes is vitamin C. At any rate, L-ascorbic acid, if present, can be readily eliminated by shaking the solution with Norit and filtering before adding the ammonium molybdate reagent. Even glycerol which may be present in syrups does not interfere.

10

M.

Experimental

2. BARAKAT

AND

S. K.

SHEHAB

Error

Some recovery experiments were carried out in which the ferrous iron added to two samplesof ferrous iron solution was in amounts differing from the amount originally present. The original sample content was estimated by means of the ammonium molybdate reagent, and the recovered ferrous iron determined by the ammonium molybdate reagent was expressedin terms of per cent recovery (Table 2). TABLE

PER CENT RECOVERY

Sample NO. 1

2

Original content Fe+ +/Zml (CLg) 74

37

Added

2

OF ADDED FERROUS

Fe+ f

IRON

Recovery of added Fe+ f

(I%)

(Ia)

300

297

200 100

204 98

Per cent recovery 99 102 98

300

302

100.67

200

200 99

100

100

99

DISCUSSION

The advanced method is based on the fact that ferrous iron instantaneously reducesammonium molybdate in the presenceof orthophosphoric acid to molybdenum blue, giving a magnificent blue color. This is in accordance with a previously published method for the calorimetric determination of phosphorus (6) in which a fresh solution of ferrous sulfate is recommendedas reducing agent for the production of molybdenum blue. Ferric salts do not give the reaction. The intensity of the blue color is proportional to the concentration of ferrous iron present in the solution. The extreme delicacy of the reaction renders it applicable in all caseswhen minute quantities of ferrous iron are to be determined, This color reaction is analogousto the reaction of ferrous salts with phosphotungstate in alkaline medium (4). The proposed method is sensitive to determine such low concentrations as 20 yg of ferrous iron. It is simple, rapid, and sufficiently accurate, since the experimental error of added ferrous iron (Table 2) does not exceed t 27*. This method has been successfully applied on various pharmaceutical preparations, e.g., syrups and tablets giving reproducible results.

MICRODETERMISATION

OF

FERROUS

IRON

11

SUMMARY A photoelectric, calorimetric method for the microdetermination of ierrous iron is described. The estimation is carried out within limits of 0.02 to 0.5 mg of ferrous iron. The procedure is quite simple and rapid but yet shows relatively high accuracy over the proposed range. This method is also recommended for the assay of pharmaceutical preparations such as syrups and tablets. REFERENCES 1.

G. J. W., Determination of ferrous iron in pharmaceutical preparations. J. Pharm. Pharmacol. 10. 351 (1937). MORTON, C., AND HARROD, D. C., Assay of saccharated iron compounds. Quart. J. Pharm. Pharmacol. 9, 480 (1936). PAUL, R. C., AND SINGH, A., Chloramine B as a bromometric reagent. J. Indian Chem. Sac. 32, 599 (1953). RICHAUD, A., AND BIDOT. .I nen- colour reaction for ierrous salts and some applications of it. J. Pharm. Chim. 29, 230; Chem. Abstr. 3, 1775 (1909). SINGH, A., Chloramine B as a vo!umetric reagent. Res. Bull. Punjab Crnin. NO. 43, 17 (1954). SUMNER, J. B., Method ior the coiorimetric determination oi phosphorus, Science loo, 413 (1944). FERREY,

Quart.

2.

3. 4.

5. 6.