Coulometric determination of serum iron

425

COULOMETRIC

DETERMINATION

OF

SERUM

IRON*

Serum iron has been determined primarily by spectrophotometric and atomic absorption methods. Coulometric determination has not been possible because the complex serum matrix contains too many substances which could interfere with a titration. In addition, none of the existing titrations for iron are compatible with the usual methods of iron separation. A new coulometric determination for. titration of iron bound to l.lO-phenanthroline, facilitates iron’ which involves coordination of the separation and titration steps. Iron-l,lO-phcnanthroline, known as ferroin. can be quantitalively formed in the.serum matrix. Since the complex is it proved to be a good choice for the very stable and resistant to oxidation, separation of iron from serum for titration. In the present paper, coulometric titrations are applied to the determination of serum iron following separation as the ferroin complex. This investigation is part of a continuing study on the application of coulometric titrations as reference methods in clinical chemistry”*“. EXPERIMENTAL

The titration cell was equipped with two side arms which were isolated from the body of the cell by fritted-glass discs. The central compartment was made from a weighing bottle of LW. 12-ml volume. All compartments were filled with generating solution. A platinum wire was used in the central compartment for the generating anode. The auxiliary generating electrode was ;I platinum foil electrode which was placed in a side-arm. A constant current of 9.65 /tA was provided by a ChrisFeld Microcoulometric Quantalizer. Model B. A Sargent Model XV Polarograph with Microrange Extender was used to maintain a constant voltage of 1.025 V across the indicating electrodes and to monitor the current passing between them. A saturated calomel electrode in the remaining side-arm and tl platinum electrode in the central compartment were used for the indicating system.

solution

The generating with cerium(

* T;kcn

in part

solution was III) sulfate.

liiorn tllo Ph.D.

Dissertation

prepared

or Sharon

by saturating

W. McClcon.

a 3 M

University

sulfuric

or Mnryhnd.

acid

1072.

S. W. M&LEAN.

426 The acid An electrolytic diluting to

acetic

W. C. PURDY

acetate buffer of pH 4.6 was prepared by combining 52 ml of 0.2 M and 48 ml of 0.2 M sodium acetate. iron solution of 0.02301 g I- ’ was made by dissolving 0.02301 g of grade iron powder in 2 ml of concentrated hydrochloric acid, and then 1 I with deionized water.

Place 0.5 ml of serum in a lo-ml cylindrical centrifuge tube with tapered bottom. Add 1 ml of 20’2; trichloroacetic acid and 1 ml of deionized water by means of an Automatic Dilutor (Lab Industries, Berkeley, California). Cover the tube with Parafilm and heat for, lo-15 min in a water bath maintained at 90-95O. After cooling, place a second Parafilm cap over the first, and centrifuge the tube for a few seconds to dislodge droplets of condensed water. Add 1 ml of chloroform and replace the Parafilm cap with a ground-glass stopper. Then shake the tube vigorously for 30-60 s. Allow built-up pressure to escape by loosening the lid. Place a Parafilm cap over the ground-glass stopper and extend down the tube for 1 in. Centrifuge at 1470 g for 10 min. After centrifugation, decant the supernatant liquid into a Buchner funnel with a sintered-glass disc of coarse porosity. Rinse the tube three times with 2 ml of deionized water delivered from a lo-ml pipet. To avoid dislodging the protein plug. allow the rinse water to flow down the sides of the tube. Use the remainder of the 10 ml to rinse the sides of the Buchner funnel, draining into a separatory funnel. To the separatory funnel, add 2 ml of 20’2; ammonium acetate. 2 ml of d.25’% hydroxylammonium chloride. 1 ml of 0.003 M 1.1 O-phenanthroline. and 2 ml of 1 M sodium perchlorate. using O-10 ml Measurcomatic Dispensers (Sargent-Welch Co.. Chicago, Illinois). Extract the contents with three l-ml portions of chloroform. To the combined chloroform extracts .in a 25-ml Erlenmeyer flask. add 1 ml of acetate buffer. and heat the flask in a 90-95” water bath for about 2 min. After evaporation of chloroform, place the contents of the flask in the titration cell, and rinse the flask with 1 ml of 4 M sulfuric acid followed by 2 ml of generating solution. Insert the electrodes and titrate at a generating current of 9.65 /LA with 1.025 V impressed across the amperometric indicating electrodes. To calculate the weight of iron in a given sample, Faraday’s Law is applied. The constant generation current is multiplied by the electrolysis time, f. This product is divided by Faraday’s constant to yield the number of equivalents of iron titrated. When this number is multiplied by the equivalent weight of iron, the result is the amount of iron present. Thus, for a current of 9.65 /lA, the weight of iron (g)=(5.5810-“)r. RESULTS

AND

DISCUSSiON

extraction of ferroin from aqueous Before serum was used, a suitable solution had to be developed. Various quantitative solvent extraction procedures for ferroin-X, were considered; X is an anion called a .“promoter” which makes possible or promotes the extraction by making the ferroin complex electrically tind the solvents’.” used with them were considered neutral. Several promoters5*’ and rejected either because of their interference with the coulometric titration or

COULOMETRY

FOR SERUM

IRON

427

the difficulty of back-extracting the complex from the organic solvent. Since perchloric acid is frequently employed% lieu of sulfuric acid for cerium titrations, it could be assumed that perchlorate ion as the promoter would not interfere with the titration. Chloroform required several extractions to remove ferroin perchlorate quantitatively from water. and upon standing for long periods, Despite these disadvantages, ferroin perchlorate precipitates from chloroform”. chloroform seemed the best solvent, since it would not affect the coulometric titration, did not form long-lasting emulsions with water, and had it relatively low boiling point (GO”). Hydroxylamine has been used to reduce iron in serum ‘.s; it will not reduce is in serum” at pH values lower than 5. Ferroin copper, a possible interference stable over the pH range 2-c). It was found that increasing the acidity of the aqueous solution decreases the extraction of ferroin perchlorate into chloroform. To decrease copper interference, to improve the extraction of ferroin perchlorate by chloroform. and to take advantage of the middle portion of the ferroin stability range, a pH of 4.0-4.5 was maintained for the extraction. This choice of pH is in keeping with the p1-l visually chosen for ferroin solvent extraction. regardless of the solvent or promoter employed. Acetate buffer is most commonly used for ferroin formation” and wits the choice here. Others have shown for iodide” and for dodecylsulfate5 that the anion : ferroin ratio should be at least 1OOO:l for quantitative extraction. That ratio was used in this work. The phenanthroline:iron ratio was always greater than 6: 1, to assure an adequate supply against possible interference. The phenanthroline: iron ratio is not critical to complex formation since this rate is reported to be extremely rapid ’ ‘. Preliminary work indicated that ferroin perchlorate returned unoxidized from chloroform to an acetate buffer of pH 4-5, if the chloroform wets evaporated by heating. Before the back-extract produced by evaporating the chloroform could be titrated quantitatively, it was necessary to determine the effect on titration times of adding acetate buffer to the titration cell. The effect was tested by performing a pretitration, and then adding various amounts of acetate buffer along with 5 Llg of iron bound to l,lO-phenanthroline. This sample was titrated. then another 5 llg of iron wits added and titrated. From these tests it appeared that the two mrr_jor factors influencing titration time are ++anges in hydrogen-ion concentration and in solution volume. The former caus9 s r;’decrease in titration time, the latter an increase. Although the two effects might to a large extent be cancelled by the use of a blank, it seemed wise to make an acetate buffer solution of ferroin acidic enough, before its introduction into the titration cell. to produce a normal time without reliance on a blank. Therefore, two different procedures for handling the sample were used. The alternative procedure involved adding 1 ml of 4 M sulfuric acid to the 1 ml of acetate buffer containing ferroin extracted from chloroform; 1 ml of this 2-ml mixture was introduced into the titration cell. The recommended procedure consists of emptying the acetate buffer into the cell, then rinsing the buffer container first with 1 ml of 4 M sulfuric acid, and finally with 2 ml of generating solution. All of the original sample is titrated in this procedure.

S. W. McCLE-AN. W. C. PURDY

428

The first procedure titrates only one-half of the original sample. and relies on buffer is not diminished by the assumption that the volume of the acetate evaporation of the chloroform. The second procedure titrates the entire sample but suffers from several defects. The increase in solution volume is 50’;.{,. four times that of the first procedure. This increase in solution volume and reagents used raises the blank titration time about 25 s to 150 s for a IO-/tA generating current. If one were titrating amounts of iron larger than 5 ccg, a larger cell could be employed and the problem of volume change lessened. if not eliminated. TABLE

I

RECOVERY OF IRON FROM WATER __________.__ ..__.. -. -...-..___ _...-._.---..-. _._ _ __ ._ ___ _^. Rlw~lxJ~I’ Pwcc’tllrw Fc Fe tctko1 .liwru/ (“4,) .

( Kl) ( IV/ ) _._______^_...__________._. -..- .____ - _....-. _._..___________....... A. With acctatc 1.003

I.150

.._ __.._.

- .._. ._. S’ I iw )

_..- ...-..- ._...._ - --

buffer

0.96 I 1.13.5

96.3 9x.7

B. With protcin-free liltroth reagents 06.2 1.180 1.135 1.10~ 1.180 100.X ____.._ _.._ __.__.____ .._, .._ ___.._._ ~_.___.._ _.____ ..__-. I’ Standard deviations wcrc calculated on trials wcrc used.

Altcrn3tivc ‘ Altcrnntivo

0.067 0.0’0

Altcrn~itivc L 0.090 I~cconimcndcd 0.075 .-...... -- .-- .-.--- ----- ------ -- IIIC basis of’ 3-7 trials. In most

instances.

more

thnn 5

The recoveries for iron added to deionized water by the alternative procedure are shown in Table I (Part A). The recommended procedure was not employed until some reagent changes had been made to accommodate the special requirements of serum. Iron was added either us ferroin or iron( III) standard. Hydroxylamine and l.lO-phenanthroline were added with both standards. Blank samples contained all the reagents. but not iron. Since copper is present in serum at about the same amount as iron, and can also form phenanthroline complexes. it was necessary to check for possible complex such as copper interference. Competition from an easily dissociated copper acetate in deionized water could be expected to be much greater than from protein-bound copper in serum. Samples containing I-/cg amounts of iron and copper were prepared in deionized water, and these samples were carried through The recovery of iron indicated no significant copper the entire procedure. interference. Henry” has reported that protein precipitation can be accomplished by vigorously shaking serum with cold chloroform for about 15 min. The top layer is the protein-free filtrate. the middle layer is thechloroform-protein gel, and the bottom layer contains excess of chloroform. Since chloroform was to be used for extracting iron from serum, it was necessary to choose a protein-precipitating procedure which used chloroform, to avoid any further precipitation during the extraction step. The methods of Caraway”, Ramsay’3. and Trinderls for disruption of the iron-protein complex and precipitation of protein were investigated. Caraway’s

COULOMETRY

FOR

SERUM

429

IRON

method uses chloroform and trichloroacetic acid at room temperature to precipitate proteins. In Ramsay’s method, the pH is lowered to 5 and the Serum is heated is added and the mixture for 5 min in boiling water: after cooling. chloroform shaken before centrifugation. Both methods have the advantage of being reasonably quick and simple, but they were found unsuitable for this work. When the supernatant, liquid from either one was shaken with chloroform. an emulsion formed which did not break even on long standing. The protein-precipit’ation procedure used in Trinder’s method for serum iron involves heating serum with 20,,,, “‘l’trichloracetic acid at 90-95” ‘for 15 min. The serum is centrifuged after cooling and supernatant liquid decanted. No chloroform is used. When this supernatant liquid was shaken with chloroform, an emulsion formed making solvent extraction unworkable. It was decided to try shaking the serum with chloroform after it had cooled and before centrifugation. Centrifugation then layer, a protein plug, and a protein-free produced three layers: a chloroform supernatant liquid. When this supernatant liquid was shaken with chloroform. only a very slight emulsion wiLs seen: it usually broke within a minute or two. With this modification, Trinder’s method for protein precipitation was much better than the other two methods tested. Ammonium acetate is usually employed in serum iron determinations to adjust the pH of acidic protein-free filtrates before color development. This compound was used in this study. Table I (Part B) shows recoveries of iron from water samples employing all the reagents which would be involved in the serum determination, including trichloroacetic acid and ammonium acetate. The control sera used for this work were from Versatol (Warner-Chilcott, Morris Plains, New Jersey) lot number 2455 121. The amount of iron present in the control sera was determined by a modification” of the method of Young and Hicks’“. Recovery data for the coulometric titrations are based on this value. Recovery studies were also made on control sera to which extra iron was added. The addition. in the form of an iron( III) solution. was made to the serum sample before protein precipitation. The amount of iron added was within the normal iron-binding capacity of serum, The results of these recovery studies arc shown in Table II. TABLE

II

RECOVERY

OF IRON

FROM

CONTROL

SERUM

A. Boscd on Babson and Klcinman method (ref. IS) 0.5 0.59 0.60 102 0.5 0.59 0.57 97 I.0 1.18 I.30 II0 B. Boscd on iron added to control serum 0.5 I.15 I.13 97 0.5 0.47 0.4K I03 “ Standard dcviutions were calculntud on the basis of 3-7 were used.

0.10 0.05 0.04

Alternntivc Recommcndcd Recommcndcd

0.03 0.04

Altcrnntivc Recommcndcd

trials. In most instances.

more than 5 trials

S. W. M&LEAN.

430

W. C. PURDY

SUMMARY

The iron in 500 141 of serum is determined by ;I coulometric titration developed for ferroin. The titration step is preceded by chloroform extraction from a protein-free liltrate of serum iron as ferroin perchlorate. Evaporation of chloroform in the presence of an acetate buffer of pH 4.6 causes the ferroin perchlorate to back-extract into the aqueous layer. The ferroin is introduced into a titration cell and titrated with electrogenerated cerium( IV). a modified amperometric end-point detection system being used.

Le dosa_ee de fer duns le s&rum (500 ~(1) cst dktermini: par un titration mCtriclue dcveloppt: pour d&ermination de fcrroin. Le titrage est pr&edC par tion du perchlorate de ferroin dans le chloroforme. Le solvant est &vapor6 d ainsi le pa-chlorate rentre duns la phase aqueuse. On fait usage de electrogt2ncrti comme titrant.

couloextracpH 4.6, Ce(IV)

ZUSAMMENFASSUNG

Eisen in 500 jtl Serum wird durch cinc fiir Ferroin entwickelte coulometrische Titration bestimmt. Vor der Titration wird das Eisen aus einem proteinfreien Serumfiltrat mit Chloroform als Ferroinperchlorat extrahiert. Durch Abdampfen des Chloroforms in Gegenwart eines Acetatpuffers von pH 4.6 wird das Ferroinperchlorat in die wiissrige Schicht zuriickextrahiert. Nacll iSberfiihrung in eine Titrationszelle wird das Ferroin mit elektrochemisch erzeugten Cer( IV) titriert. wobei ein modifiziertes amperometrisches System fir die Bestimmung des Endpunktes verwendet wird. REFERENCES 1 S. W. McClcmn

and

W. C. Purdy.

2 R. J. Troy and W. C. Purdy. 3 4 5 6 7 X 9 10 1I I2 I3 14 IS I6

C/h.

rlw/. C/I~III. Acvtr. 67 ( 197.1) C/rim .Icttr. 27 ( 1970) 401. C/h. C/IWI.. IX ( 1972) 503.

I 13.

M. A. Brooks and W. C. Purdy. F. Vydra and R. Pribil. Ttrhrcc. 3 ( 1959) 72. R. Powell and C. G. Taylor. C/ro~r. /UC/.. ( 1954) 726. C. W. Margcrum and c’. V. Banks. .4rw/. ~‘hw.. 26 ( 1954) 200. W. N. M. Ramsay. Uiochw. J.. 53 ( 1953) 227. H. L. Williams and M. E. Conrxl. J. Ltrh. C/h. hld. 67 ( 1966) I7 I. A. A. Schilt. /I r~tr/_~~fictrl Applictrfims 01’ J./O-Plrc~ticrrrrh~c~lilrc~ trrrd Rcltrrctl Cor~rp~rr~tls. New York. 1’369. p. 6 I. I. M. Koltlwl’f. T. S. Lee md D. L. Lcussing. ,drw/. Clrrrr~.. 20 (1948) 9x5. R. J. Henry. Clirriccrl C/t~v~~i~tt’~: Harper nnd Row. New York. 1966. lx 170. W. T. Curavmy. Clirr. Chcrrr.. 9 ( 1963) 188. W. N. M. Rummy. C/h. Clriw. Acrtr. 2 ( 1957) 2 14. P. Trindcr. .I. Clirr. Pc~r/ro/.. 9 ( 1956) 170. A. Babson and N. Klcinman. C/in. C'/IC~.. I3 ( 1967) 163. D. Young :~nd J. Hicks. J. Clirr. Ptrr/~o/.. IX ( 1965) 98.

Pcrgtmon

Press.