1216
ANALYTICAL CHEMISTRY
this mc~tliotlcalled for the addition of amnionium chloride, eliminating the use of a chloride balance for cobalt. Preliminary qualitative work showed that both nickel and cobalt are slo\vly precipitated from neutral solutions by ammonium oxalate. These precipitates dissolve upon the addition of ammonia. Such ammoniacal solutions remain clear when esposed to the atmosphere for one week. After this period, some nickel salt separates from solution, presumably through the loss of ammonia, for the precipitate redissolves upon the addition of more ammonia to the solution. T h e procedure was checked by eniployiiig it for the analysis of fivr synthetic mixtures of calcium, nickel. and cobalt chlorides in varying proportions, prepared from stork solutions of pure salts. The results are presented in Tahle I, a h i r h indicates tht. method to
be reliable and free from systematic error. The average error for each component borders on the limit of accuracy of the analytical technique used for its determination in the stock solutions. LITERATURE CITED
S.,and Damerell, 1'. R., "Quantitative Analysis," pp. 174-7, New York, McGraw-Hill Book Co., 1940. ( 2 ) Garwin, Leo. and Hixson, A. N., Ind. Eng. Cheni.,41, 2298, 2303 (1949). (3) Hillehraiid, IT. I;., arid Lundell, G . E. F., "Applied Inorganic .\iial>.sis." p. 315, S e w York, John Wiley & Sons, 1929. (4) Kuhl. G. IT-., Chem.-Ztg., 53,279 (1929). ( 5 ) Willard, H. H., and Diehl, Harvey. "Advanced Quantitative Analysis," p. 384. New York. D. Yan Sostrand Co., 1943. (1) Booth. H.
R ~ ; r s : r ~ i.Iiiiir : ~ , 2 8 . 1948.
Colorimetric Determination of Serum lron FK LVCES JONES,
Haylor I'nioer.*ify,Dallas, Tex.
i r o n is determined colorimetricall) as the complex formed with thioc) anate. To increase the specific color intensit) of the solution to be measured, the iron complex is extracted and concentrated in ether. The ether extract is measured at 500 mp, where its absorption is greatest. For the anal>sis of serum iron, nitric acid is used to precipitate proteins, to split off iron bound to proteins, and to oxidize ferrous iron. S o digestion is necessar?. Uthough the method is designed for determination of serum iron, it ma! he used for the estimation of small amounts of iron in other soltctions.
-1SY invrstigators including JT-ong ( I O ) , llooi,c~rt a i . (8), Rarkan ( I ) , ant1 sewral others (2-4, 6, 7 , 9) h a w dvveloped colorimetric mr,thods for t h r determination of st'runi iron, utilizing the colorc4 conip1c.s foriiit~din t h r relaction of f w i c iron with thiocyanate,
In the \\.ong method, oi~ganic.imn is released tiy oxidation with sulfuric acid and potassium persulfate. The proteins are prrcipitated with tungstic acid. Thiocyanate is added to the filtratci :ind the iron is deterniinrJ colorimetrically. The low estinetion coefficient for small amounts of F e S C 1 - I in aqueous solution and the relatively high reagent blank found in this procedure make serum iron determinations resulting from this method subject to i,:ither large errors. In the Moore method the serum is digested with sulfuric acid :md hydrogen peroxide. The osidized iron is then treated with thiocyanate and the iron complex thus formed is extracted with isoamyl alcohol. The higher estinction coefficient of the isoamyl alcohol solution of the iron comples improves the accuracy of the determination over the \Tong method. HoLvever, t h e technique requires large amounts of serum ( 5 t o 10 ml.) and the acid digestion procedure is time-consuming. In th; Barkan method, which can use as little as 1.5 nil. of serum, 'peroxide" ether is used as a solvent for the thiocyanate color. The use of the unstable "peroxide" promises certain tiis: d v : i n t q e s in routine use in many laboratories.
sepuatorj. funnel and 1.6 nil. of :3 .Y potassium thiocyanate are added. The misture is shaken for 1 minute with about 3 volumes of peroxide-fi,er ether which has been saturated with water, and the ether layer is seporated. This extraction is repeated twice, and the three ether fractions are combined, evaporated to a small volume, anti niade u p qu:intit:itively to 5 nil. with ether. €3ec:iusr
0.80
0.60
u.
0.40
In thti investigation described here an attempt was made to devt.lop an accurate and rapid mt,thod for the estimation of srnall amounts of serum iron. S i t r i c acid is used to precipitate wruni proteins, to split off protein-bound iron, and to oxidize any f(1rrous iron present. T o the ferric iron thus made availabk potassium thiocyanate is added. The iron complex is thtln extractcd with c,thcr and concentrated beforr being measurcxd c~oloi~inic~trically. PROCEDURE
Clear, hemoglobin-free serum (1.5 ml.) is diluted with 4.5 nil. of distilled water and acidified with 6 mi. of 1 JI nitric acid. The niisture is allowed to stand for at least 5 minutes for complete precipitation, is then centrifuged and the precipitate is discarded. .\n 5-ml. aliquot of the clear supernatant is poured into a 60-nil.
0.90
t
01
1
450
Figure 1.
I
I
I
490 WAVE LENGTH, M P
530
Spectral Absorption of Iron Thiocyanate Complex in Ether and in Water
1217
V O L U M E 21, N O . 10, O C T O B E R 1 9 4 9 of the easy volatilization of t h e ether, a n y convenient vessel may h e used for the concentration; care should be taken t o avoid loss by entrainment. Final adjustment of the volume may be made in a tube graduated t o t h e precision required. For many purposes a IO-ml. graduated pipet or even R graduated centrifuge tube is ant isfactory.
chloroform were tested for the cktraction of thiocyanate iron v o m plrs. Of those tested, ether gave the most complete extraction and the highest absorption for the iron complex. The intensity ot t h r iron complex color was found to be stable for several hours. To ulirck the linearitv of the color reactions, the extinction VI)cficirnts of standard samples of varying concentrations were detrrniined. T h a t Bwr's law applies over a useful concmtrxtion rang. for +rum iron is sho\vn in Figure 2
""n eo
I . Comparison of Sitric Acid-Ether Extraction >\lethod and Moore lIethod for Determination of Seriini
Table
Iron sa!! I[ >It.
Nitric dcidEther Extraction Method --a,
87 2 Lz
30
I-
"I-
L
fi
'OI
l I u o r r JIetliod
1
o/ ii,o,i i n 190 rn/.--
84.6 86.8 98.4 102.3 88.fi
89.7
85 i 85 6 87 3 97 8 '19 6 89 8 88 t i
+I 7 -1 0 -0..i +O.$ f2.I
-1.2
-1.1
2 0
I O
;
140
200
30 0
CONCENTRATION OF IRON ( M I C R O G R A M S PER MILLILITER) Fipirre 2.
E x t i n c t i o n Coefficients of Standard Saniples
,.1
iron c.oiicentrittioii is then detemiirietl l)y r o n i p ~ ~ i i it hc, g ii standard iron solution treutcd i n tht. \v:iy. I n the tr:msi'er anti handling of the ether solutioii :iftt,r adjuqtment to volumc,, c ire must be talicw to :tvoid l a q v p i i t , Curett cs u s c ~ fl o i . nic~:iwi~ing tic,
roloi. intrnsity with that of nit^
To tlt~tt~rmine thc conipletcncss oi' thc extraction of th(3 i w i i compl(~swith ether, tht: partition coefficient for the iron thiocy:inatcx complex betn-ecn tsther and water was experimentally dctc~i.iiiiriedand found to brl 8.09 at 28" c'. Three ether extractions riduccld thtx iron in th(1 \vater wsiduc to a negligible amount. I \ - h t b n tht. iron concentration is ~ O T T t, h r largc volumo of coniparativoly dilute ether solution thus obtained may ncw&tatc~ (,vaporation to conccsntratc the iron coniples for mort accuratt' , S o change \\-a? found i n the final iron valucs of analyxtd which u-crc rend btlfore tlvaporation and aftcii c~vaporation. A s in any method for tht, detcxi.mination of serum iron, this pi'occdulc is liascd on thv us(%of iron-free reagents and ht,nioglohinr u m , II-hcn hi,moglobin is present in thv wruni, a correct i o i i rnay i i i ' made 1)y inclasuring the atxorption a t 576 nip and cx1cul;iting thc' lic~nioglol~in iron. (Ilcmoglobin is 0.33y0iron, 6 ) .
EXI'ERIIMEYTAL
.\I(~irsurc*nieiits for thi5 qtudy w i r e inad(. ivith a l3eckman D.C. sr",cti,oi'hotonirtc.I. a t 500 nip, using thcs blurx-sensitive photocell and a slit width of 0.04 mm. IIi~\vcver,with a filter of the propci' spt'ctral characteristics ( s u c h its the It-ratten S o . 75 filtctr) any colorinit~toi~ may lw uscd. S o normall>- occurring constituwit of wi'uni othcr than iron iurnishrs an ether-extractahlc compound in t h v proccdurcs cltwrihc~rlwhich has an absorption band in the nicasui,cd range. I'.w of t h r mvthod should not be limitcd t o dcbtc.i,minatioii of' ,wruiii ii.on but should hc applicabl(~to oth(5r solutions containing small :iniouiitb of iron. Thca st'ruiii iimi in t(w ,sanip1t~~ of normal s ( w m was determined both t h r method of l l o o r c ~and the method outlined her(>. Thfh rosulti; listed in TaI)l(, I slio\v good agrrschnicnt between this t \ v o nicthotis. l i c ~ o v c ~ rcsprrimcnt y s \ v t w run i n Ivhich iron \vas addrd t o f i v c x aiialyzcd samples o f wruni. In this st't of analysis, 1 nil. of 11 ,standard iron solution (100 niicrogranis p
