Water-Soluble Sulfonated Chromogenic Reagents of the Ferroin Type and Determination of Iron and Copper in Water, Blood Serum, and Beer with the Tetraammonium Salt of 2,4-Bis(5,6Acid diphenyl- 1,2,4-triazin-3-yl)pyridinetetrasulfonic George L. Traister and Alfred A. Schllt" Department of Chemistry, Northern Illinois University, DeKalb, 111. 60 115
Four highly sensitive chromogenlc reagents of the ferroin type were sulfonated to provide more useful, water-soluble derivatives. The desiredderivatives were isolated in ammonium salt form, identifled by elemental analysis and spectral studies, and characterized as chromogenic reagents for Iron and copper. The most sensitive of the four, indeed the most sensitive iron(ll) chromogen reported to date of the ferroin type, proved to be the tetraammonium salt of 2,4-bis[5,6-bis(4-phenylsulfonic acid)-1,2,4-triarln-3-yl]pyridine (2,4-BDTPS). Applied to the simultaneous determination of iron and copper in blood serum and in potable water, 2,4-BDTPS proved highly sensitive, yielding results with precision and accuracy characteristic of good spectrophotometric procedures. Methods utillzlng 2,4BDTPS for the selective determinationof iron in blood serum and in beer also proved highly effectlve and sensitive for 1 p or less of iron.
Systematic studies by Smith and co-workers of ferroin-type compounds, specially synthesized by Case and his students, have produced a variety of outstanding chromogenic reagents for iron and copper (1-4). From their joint program of research, Smith and Case have provided also important guidelines for design of special purpose reagents. An important example is their discovery that phenyl substituents, in positions para to the ferroin nitrogen atoms, greatly enhance the molar absorptivities of the metal chelates. This principle prompted Case to synthesize over the ensuing years such notably sensitive metal chromogenic reagents as bathocuproine ( 2 ) , terrosite (5), P P D T (6),and P D T (7). These phenyl substituted compounds, because of extremely limited water solubility, are best employed in conjunction with an extraction procedure to preconcentrate and isolate trace amounts of copper and iron for subsequent spectrophotometric determination. They afford the added advantage of enabling prior removal of these same metal ions from reagents by solvent extraction thereby greatly minimizing reagent blanks. Unfortunately, because of the water insolubility of these reagents and their metal complexes, they are unsuited for special applications requiring speed and simplicity, for example in automated procedures and in routine analysis of large numbers of samples. A means of overcoming the solubility limitation was first devised by Trinder who treated bathophenanthroline with chlorosulfonic acid to provide a water-soluble sulfonated derivative (8).Subsequent studies by Zak, Diehl, and others provided sulfonated derivatives, some isolated in solid form, of bathophenanthroline (9, IO), bathocuproine (IO),P D T ( I I ) , and terrosite (12,13).All retain their special chromogenic advantages after sulfonation but with the added feature of high solubility in aqueous solutions. Their applications in analysis of blood serum and water for copper and iron are particularly noteworthy for speed, simplicity, and sensitivity (9-14). 1216
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 19713
More recent syntheses by Case have provided a number of new ferroin-type compounds with phenyl substituents and high sensitivities as reagents for iron (15, 16). One of these has proved to be the most sensitive iron(I1) chromogen reported to date: 2,4-bis(5,6-diphenyl-1,2,4-triazin-3-yl)pyridine, hereafter referred to as 2,4-BDTP. In chelation of iron(I1) it acts as a bidentate ligand to form an intensely magenta colored tris chelate, extractable into chloroform, with a molar absorptivity of 32 000 at 563 nm. Neither 2,4-BDTP nor its metal chelates are soluble in aqueous or alcoholic solutions; hence, a chloroform extraction procedure is necessary for successful application to spectrophotometric determinations. We therefore undertook preparation of a water-soluble sulfonated derivative of 2,4-BDTP. As an integral part of the investigation, we also included attempted sulfonations of three other phenyl substituted triazine derivatives: 2,6-bis(5,6-diphenyl-1,2,4-triazin-3-yl)pyridine (2,6-BDTP); 3-(4-phenyl-2pyridyl)-5,6-diphenyl-1,2,4-triazine (PPDT) and 3-(2-pyridyl)-5-phenyl-1,2,4-triazine (PPT).The results are reported here together with descriptions of several practical analytical applications for the most sensitive chromogen of those prepared, the sulfonated derivative of 2,4-BDTP, which will be referred to as 2,4-BDTPS (see Figure 1).Reference to the other sulfonated compounds will be made using the Roman numeral assigned each (see Table I).
EXPERIMENTAL Reagents. The 3-(2-pyridyl)-5-phenyl-1,2,4-triazine was synthesized as described by Culbertson and Parr (17),and the 2,6-bis(5,6diphenyl-1,2,4-triazin-3-yl)pyridine was prepared as reported by Case (15). Samples of PPDT and 2,4-BDTP were obtained from the G . Frederick Smith Chemical Company (Item Nos. 661 and 663, respectively). A 0.005 M solution of 2,4-BDTPS (G. Frederick Smith Chemical Co., Item No. 664) was prepared by dissolving 0.48 g of 2,4-bis(5,6diphenyl-1,2,4-triazin-3-yl)pyridinetetrasulfonic acid tetrasodium salt (2,4-BDTPS) in 100 ml of distilled water. Standard solutions of iron and copper were prepared in known concentrations from weighed amounts of electrolytic metals. A 10%solution of hydroxylamine hydrochloride was prepared by dissolving 100 gin 900 ml of distilled water. Traces of iron and copper were removed by treatment with excess PDT followed by extraction with isoamyl alcohol (7). Special reagent solutions were prepared for analysis of blood sera. The extractant-reductant solution was 0.2 M in hydrochloric acid and contained 0.5% hydroxylamine hydrochloride (free of iron and copper). The protein precipitant solution was prepared by mixing 12 ml of trichloroacetic acid with 88 ml of distilled water. The special acetate buffer solution was 0.3 M in ammonium acetate and of sufficient ammonia concentration (approximately 2.5 M) so that, when added to the treated serum sample, the final solution would have a pH between 5 and 6. The special tartrate buffer was l M disodium tartrate, prepared to produce a pH between 2.5 and 3.5 when added to the serum sample treated with protein precipitant solution. All other chemicals were reagent grade. Apparatus. Spectra were recorded in the visible region with a Cary Model 14 recording spectrophotometer and in the infrared region with a Perkin-Elmer 237B grating infrared spectrophotometer. Proton
Table I. Elemental Analysis of Sulfonated Compounds Found, %
Calcd, % Compounds
IA IB I1
'I1 IV
Formula
C
C,,H,,N7(S03)4(NH,)4.2H20 43.5 C3,H,,N,(SO3),(Na),.4H,O 41.1 C3,H,,N,(SO3)4("4)4 *2H2O C26H18N4(S03)3("4)3'H20 C14H10N4(S03)(NH4)'H20
43.5 44.9 48.1
H
N
3.97 2.64 3.97 4.17 4.30
16.0 9.6 16.0 14.1 20.1
S
13.3 12.5
...
... ...
C
H
43.2 40.3 43.5 45.1 48.4
3.90 2.56 4.02 4.00 4.16
N
15.1 9.3 15.0 13.2 19.4
S
13.4 12.6
... ... ...
a Identity of compounds: IA, ammonium salt of 2,4-bis[ 5,6-bis(4-phenylsulfonicacid)-1,2,4-triazin-3-yl]pyridine; IB, sodium salt of I A ; 11, ammonium salt of 2,6-bis[ 5,6-bis(4-phenylsulfonicacid)-1,2,4-triazin-3-yl]pyridine; 111, ammonium salt of 3-(4-(4-phenylsulfonic acid)-2-pyridyl] -5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine; IV, 3-(2-pyridyl)-5-(4-phenylsulfonic acid)-1,2,4-triazine.
Figure 1. Structure of 2,4-BDTPS
magnetic resonance spectra were recorded with a Varian A-60-A NMR spectrometer. Absorbance measurements a t individual wavelengths in the visible region were also made using a Beckman Model DU spectrophotometer equipped with a photomultiplier detector. A Corning Model 7 pH meter, with saturated calomel-glass electrode system, was used for pH measurements. Elemental analysis for carbon, hydrogen, and nitrogen was performed using a Perkin-Elmer Model 240 elemental analyzer. A Parr peroxide bomb apparatus (Series 2100) was employed for decomposition of samples prior to sulfur determination. For absorbance measurements of serum iron, a 1.00-cm microcell (1-ml vol.) was used. Wet ashing was performed in 125-ml conical borosilicate glass flasks under a transite hood. Preparation and Analysis of Sulfonated Compounds. The parent compounds were sulfonated by the method of Cryberg and Diehl(20), isolated as ammonium salts, recrystallized from distilled water, and dried in air. 2,4-BDTPS was isolated also in the sodium salt form. All of the sulfonated products were light yellow, water soluble solids. Carbon, hydrogen, and nitrogen determinations were performed by J. Darby. Sulfur contents of the sodium salt and of the ammonium salt form of 2,4-BDTPS (IA and IB in Table I) were determined after decomposition of the samples in a Parr bomb by a gravimetric procedure (22). Infrared spectra were obtained of the samples in KBr disks. Deuterium oxide was used as solvent for the sulfonated compounds, in recording NMR spectra. Simultaneous Determination of Iron and Copper in Blood Serum. Pipet 0.50 ml of serum sample into a 13 X 100 mm test tube, add 0.50 ml of extractant-reductant solution, mix, and let stand approximately 10 min. Add 0.50 ml of protein precipitant solution, mix, stopper the tube, and centrifuge until supernatant is clear. Transfer 1.00 ml of the supernatant to a second tube, add 0.30 ml of 0.005 M 2,4-BDTPS and 0.20 ml of the special acetate buffer solution (to adjust to pH 5-6), mix well, and allow to stand for 10 min for maximum color development.Measure the absorbance at 563 and also at 460 nm. Carry a reagent blank through the procedure utilizing 0.50 ml of distilled water in place of the sample. Subtract the absorbances of the blank from those of the sample. From the corrected absorbances of the sample, calculate the concentrations of each metal by simultaneous solution of the two linear equations, coefficients for which having been determined by carrying standards through the same procedure as unknowns (18).For convenience and simplicity a suitably prepared monograph can serve effectively in place of the calculations (19). Selective Determination of Iron i n Blood Serum. Follow the same procedure as the above (for the simultaneous determination of iron and copper) except in place of the 0.20 ml of the acetate buffer, add 0.20 ml of the special tartrate buffer to adjust the pH to 2.5-3.5. Heat the mixture for 15 min at 40 "C, cool, and measure the absorbance at 563 nm. Carry a reagent blank and standard through the same procedure. Calculate the concentration of iron in the unknown from the measured absorbance corrected for the blank or refer to a suitably prepared calibration curve.
Determination of Iron and Copper in Water. Pipet a sample of appropriate size (1-20 ml containing 4-40 fig of Fe and/or 16-160 fig of Cu) into a 50-ml conical flask,add 3 drops of 6 M hydrochloricacid, and bring to near boiling on a hot plate. Cool and transfer the contents of the flask quantitatively to a 25-ml volumetricflask. If only dissolved iron, free of particulate matter, is to be determined, the sample need not be acidified and heated but can he delivered directly into the 25-ml volumetric flask. Add 1ml of 0.005 M 2,4-BDTPS and 1ml of 10% hydroxylamine hydrochloride solution, mix thoroughly, add 1 ml of 10 M ammonium acetate (to adjust to pH 5-7), and dilute to volume with distilled water. Measure the absorbance a t 563 and also a t 460 nm. Carry a reagent blank and standard through the same procedure to obtain suitable corrections and calibration data. Calculate the concentration of each metal (18)or determine them from a suitable monograph (19). As an alternate method (7) for the simultaneous determination, measure the absorbance a t 463 nm before and 1-15 min after adding 10-30 mg of sodium cyanide to the contents of the flask. The iron concentration is directly proportional to the final absorbance, and the decrease in absorbance on adding sodium cyanide is linear with copper concentration. Calibration curves are simpler to prepare and use in this than the above method. Determination of Iron in Beer. Pipet a 25-ml sample of degassed beer into a 125-ml flask and evaporate to near dryness on a hot plate. Cool and add 5 ml of an equal volume mixture of concentrated nitric acid (70%)and perchloric acid (68%).Heat gently until dark brown fumes cease to evolve. Continue heating at a higher temperature until the solution becomes colorless and dense white fumes of perchloric acid fill the flask. To the cooled flask, add 5 ml of distilled water, 1ml of 0.005 M 2,4-BDTPS, 2 ml of 10%hydroxylamine hydrochloride, and 3 ml of 1M disodium tartrate. Adjust the solution to pH 2.5-3.1 with concentrated ammonium hydroxide (requires approximately 0.5 ml). Heat the solution at 40-60 OC for 15 min to produce complete color development. Cool to room temperature, transfer the solution quantitatively to a 25-ml volumetric flask, and dilute to volume with distilled water. Measure the absorbance at 563 nm. Carry a reagent blank and standards through the same procedure. Make use of a suitably prepared calibration curve or empirical equation to convert absorbance to concentration. Interference Study. Solutions prepared to contain 1.20 ppm iron and known concentrations of various substances were analyzed for iron content by the following procedure. Pipet a 5-ml sample into a 50-ml volumetric flask; add 1ml of 0.005 M 2,4-BDTPS, 1ml of 10% hydroxylamine hydrochloride, and 2 ml of 10 M ammonium acetate; dilute to volume with distilled water, and measure the absorbance at 563 nm. An absorbance differing from the expected volume by 3% or greater was assumed to be indicative of interference by the added substance.
RESULTS AND DISCUSSION Identification of Sulfonated Products. The results of elemental analysis, compiled in Table I, a r e consistent with the expectation that each a n d every phenyl substitutent is monosulfonated. Good agreement between calculated a n d experimental values for carbon a n d hydrogen indicate that t h e products are reasonably pure, in spite of t h e considerable difficulty experienced in their isolation a n d a t t e m p t e d purification. Results for nitrogen a r e consistently low for t h e ammonium salts but not appreciably low for IB (the sodium salt of IA), probably d u e t o incomplete neutralization of t h e sulfonic acid groups and/or loss of ammonia by hydrolysis and volatilization i n the course of recrystallization from water. ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1217
Table 11. Visible Absorption Maxima of Iron(I1) and Copper(1) Chelates of Sulfonated and Unsulfonated Compounds Iron(I1)
Copper(1).
Sulfonated Compound
A , nrn
Unsulfonated A, nm
E
Sulfonated E
565 32 200 563a 32 OOOa 470 18 200 468b 18 OOOb 580b 14 OOOb 580 1 4 100 30 700 561b 28 700b I11 563 555b 28 1 O O b IV 553 28 700 a Chloroform-ethanol solutions; values from Ref. ( 1 6 ) .b Ethanol -water only slightly different from that of 11. I I1
A, nm
E
A, nm
f
460 c
9 700 c
510a
1 5 200a c
I
I
500
6 00
I
WAVELENGTH, nm
C
463 5 200 480b 7 900b 460 5 100 480b 9 500b solutions. C No appreciable absorption; spectrum I
I
Unsulfonated
2
I
I
I
I
I
I
I
I
6
4
1
I
I
1
8
PH
Figure 2. Absorption spectra of metal chelates of 2,4-BDTPS and of reagent blank vs. air: Curve A, blank; Curve E, 2.96 X lod5 M Cu(l); Curve C, 1.46 X M Fe(ll)
Figure 3. Effect of pH on completeness of color formation with 24BDTPS: Curve A, 2.16 X M Fe(ll)at 563 nm; Curve E, 4.89 X M Cu(l) at 460 nm
Isolation of products in the sodium salt form would have been preferable in this regard; however, neutralization with ammonia was selected in preference to sodium hydroxide because excess ammonia can be readily removed by boiling, also ammonia is less contaminated by trace metal impurities. Infrared spectra, omitted here for brevity, confirm that each of the four compounds was successfully sulfonated, as evidenced by a broad absorption band in the 1175-1200 and a narrower but strong band in the 1025-1030 cm-l region. Furthermore, each compound displayed a spectral pattern in the hydrogen out-of-plane bending region (700-900 cm-l) characteristic of para-disubstituted benzene. In each case, a distinctive difference between spectra in this region of parent and sulfonated derivative is the presence of an intense band in the 800-860 cm-l region for the sulfonated compound. This lends support to the assumption, based on synthetic organic expectations ( 2 2 ) ,that each phenyl substituent is mono-sulfonated in the para position. Specific assignments for the proton magnetic resonance spectra of the sulfonated and parent compounds, because of the complexities of multiple spin-spin interactions, proved too difficult for us to accomplish without recourse to spin decoupling and model compounds. The salient features of the spectra, however, at least appear to be consistent with a model in which each phenyl group is sulfonated (as indicated by the down-field shift of a multiplet believed to be that of the phenyl protons) and each sulfonic group is in the para position (as suggested by apparent lack of increased multiplicity which should have resulted for ortho or meta sulfonation). Visible Absorption Characteristics of Metal Chelates. Wavelengths of maximum absorbance and corresponding molar absorptivities of the iron(I1) and copper(1) chelates are listed in Table I1 together with those of the unsulfonated analogues. Comparisons reveal that sulfonation does not impair the sensitivities of the compounds as iron chromogens.
The spectral characteristics of the iron(I1) chelates remain essentially unchanged. Spectra of the sulfonated copper(1) chelates, however, differ appreciably from those of the unsulfonated complexes. Sulfonation gives rise to diminished molar absorptivities and sensitivities for copper(1). Similar effects are seen in the results obtained by Blair and Diehl for bathophenanthroline and bathocuproine (10).Further study would be of interest to learn if these effects are due to solvent, electronic, steric, or a combination of influences. Of the four new water-soluble chromogens, 2,4-BDTPS is the most sensitive for iron and for copper. Spectra of the iron(I1) and copper(1) chelates and that of the reagent blank, shown in Figure 2, are sufficiently different to enable simultaneous determination of the two metals. It is evident that the two most suitable wavelengths for this purpose are 460 and 563 nm, although, for special purposes, wavelengths of 563 and 600 nm could be used to avoid spectral interferences from other species that absorb at 460 nm. Absorbances of the iron(I1) and copper(1) chelates follow Beer’s law and are additive, permitting simultaneous determinations to be made. Based upon the respective molar absorptivities and an absorbance detection limit of 0.005 in a 1-cm cell, an iron concentration as low as 1.6 X lo-’ M (0.009 ppm of Fe) and copper at a concentration of 5 X M (0.033 ppm of Cu) can be detected with 2,4-BDTPS. The chromogenic reactions of 2,4-BDTPS with metal ions other than copper and iron were also investigated in light of their possible interferences. Cobalt(I1) forms a pale orange chelate in solutions of pH 5 to 8 which exhibits a broad shoulder in its visible spectrum at 480 nm (extending from 500 to about 425 nm, where the free ligand absorbs strongly) with a molar absorptivity of 400. Similarly, nickel(I1) forms a pale yellow chelate from pH 3 to 8 that displays a broad shoulder extending from 520 to 460 nm in its spectrum, with a molar absorptivity of 380 at 480 nm. First row transition metal ions
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ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
Table 111. Effect of Various Ions on the Determination of Iron with 2,4-BDTPS No interference from 100 ppm of any of the following NH,+, Li+, Na+, K+,MgZ+,Ca2+,Srz+, BaZ+,A13+ Snz+,Pb2+ As3+ Bi3+,V4+, MnZ+,Znz+, Cd2+ F-, Cl-, Br', I-, &lo4-, BrO,-, IO3-, CO,'-, SO,z-, NO,-, NO,-, PO,3-, SCN-, acetate, trichloracetate, borate; citrate, oxalate, tartrate Interfering ions (tolerance levels in ppm) Cr3+ (21) Ni2+ (3) CN- (2) coz+(7) Cu+ (0.3) p,o,4- (2) other than iron, cobalt, nickel, and copper failed to yield appreciably colored complexes with 2,4-BDTPS. Effect of pH. The results plotted in Figure 3 show the influence of pH on chelation of iron(I1) and copper(1) based upon measurements made soon (5-10 min) after mixing (in the order cited) standard metal ion solution, reductant, buffer, and 2,4-BDTPS. Incomplete formation of the chelates a t or above pH 7 in these experiments is in part due to slow kinetics, because the absorbances gradually approached their maximum levels on prolonged standing. Above p H 9, no appreciable color formed with either iron or copper, even after long standing. Color development is rapid and complete a t pH 2 to 6 for iron(I1) and 5 to 7 for copper(1). Once formed, the iron(I1) chelate is slow to fade in color, even in solutions of pH less than 1 or greater than 11;within the pH range 2-8, it is stable for days. The copper(1) chelate is more labile and rapidly fades if its solution is made strongly acidic or basic. For solutions maintained a t pH 4 to 8, the copper(1) chelate remains stable for a t least 1 hour; however, spectra are noticeably altered after 1 day. O r d e r of Addition. Low results for iron were sometimes obtained unless 2,4-BDTPS was present prior to pH adjustment, presumably to prevent precipitation of hydrous iron oxides. Hence the recommended order of addition of reagents to acidic sample is as follows: 2,4-BDTPS, hydroxylamine hydrochloride reductant, buffer, and water to dilute to volume. Effect of Foreign Ions. Results of the interference studies are given in Table 111. As expected, positive errors in the determination of iron resulted due to the presence of modest amounts of cobalt, nickel, or copper or large amounts of chromium. Interference by pyrophosphate or cyanide ions can be eliminated by acidification with hydrochloric acid and boiling to volatilize hydrogen cyanide and hydrolyze pyrophosphate to orthophosphate. Analysis of Blood Serum. Results obtained from simultaneous determination of iron and copper in a pooled sample of human blood sera are compiled in Table IV together with data for samples spiked with iron and with copper to test recoveries. Precision for determination of iron proved superior to that for copper, primarily because the sample size was such to favor optimum precision in absorbance measurements at 563 nm. To obtain approximately the same relative precision for copper as for iron, it is estimated that the copper concentration should be approximately 4-fold greater than that of iron. Recoveries for both iron and copper proved to be quantitative, accurate to within the precision of the measurements. In effect, the method of standard addition was applied and confirmed that the results obtained for unspiked samples are reliable. Analysis of the same pooled blood sera sample by the selective determination of iron procedure yielded the results compiled in Table V. Recovery of iron from spiked samples proved quantitative, and added amounts of copper were without adverse effect. Thus, the use of tartrate ions to mask copper, in conjunction with buffering a t pH 2.5-3.5 to dis-
Table IV. Simultaneous Determination of Iron and Copper in Blood Serum with 2,4-BDTPS Cu, ppm Sample
Present
Pooled, untreated
X
X X X X X X
Pooled, iron added Pooled, copper added
X + 0.32 X + 0.78 X + 1.56 Mean of X Std dev
Fe, ppm Found
1.46 1.82 1.64 1.46 1.50 1.32 1.64 1.90 2.54 3.00 = 1.56 = 0.16
Present
Y Y
Y Y + 1.21 Y + 1.21 Y + 1.45 Y + 1.45
Y Y Y Mean of Y Std dev
Found
1.13 1.18 1.15 2.38 2.34 2.60 2.64 1.14 1.19 1.19 = 1.16 = 0.02
Table V. Selective Determination of Iron in Blood Serum with 2,4-BDTPS by pH Control with Tartrate Buffer Added, ppm Sample
Fe
Untreated
0
Treated with Fe
0 0 1.38 1.38 1.38
Treated with Cu
0 0 0 0
cu 0 0
0 0 0 0 0.31 0.79
1.57 3.92
Found Fe, pprn Total
Serum
1.16 1.16 1.13 1.13 1.17 1.17 2.52 1.14 2.64 1.26 2.50 1.12 1.14 1.14 1.11 1.11 1.19 1.19 1.16 - 1.16 X = 1.16 Std dev = 0.04
Table VI. Comparison of Serum Iron Determination by Four Different Methods Applied to the Same Pooled Blood Sample Mean Method
Simultaneous Fe and Cu Selective (tartrate buffer)
Fe, pprn
Std dev
No. of Detmns
1.17 1.16a 1.18 1.16b 1.20
0.04 0.02a 0.04 0.046 0.05
6 10 6 10 6
Sodium cyanide addition (final step)c Direct measurement 1.40 0.04 6 (no corrections)d a Data from Table V. b Data from Table VI. C Procedure same as that for the simultaneous determination method except for final step of adding sodium cyanide. d Same as simultaneous method except absorbance is measured only at 565 nm. courage chelation of copper by 2,4-BDTPS, proved very effective. The main disadvantage of the procedure is the need to heat the solutions for 15 min at 40 "C in order to effect complete formation of the magenta iron color. Apparently, iron(I1) is complexed by tartrate and/or serum components and exchange of ligands for 2,4-BDTPS, although thermodynamically favored, is slow unless the solution is heated. At the same pH and in the absence of tartrate or blood serum, complete formation of the iron(I1) chelate is rapidly attained. Results obtained for iron in the pooled sample by four different methods are compared in Table VI. All determinations, six replicate ones for each of the four methods, were completed the same day. Included also, labeled appropriately, are results obtained several weeks earlier for the same pooled sample. The first three methods listed in Table VI yielded comparable ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1219
Table VII. Determination of Iron and Copper in DeKalb Municipal Drinking Water with 2,4-BDTPS
Table VIII. Selective Determination of Iron in Beer with 2,4-BDTPS by pH Control with Tartrate Buffer Iron found. vvm
Added, ppm
Total found, p p m Sample treatment
cu
Fe
Untreated 0 1.21a Iron added, 0.43 ppm 0 1.66 Iron added, 0.96 ppm 0 2.18 Iron added, 1.44 ppm 0 2.64 Iron added, 1.93 ppm 0 3.10 Iron added, 2.40 ppm 0 3.62 Copper added, 0.31 ppm 0.38 1.20 Copper added, 0.63 ppm 0.62 1.20 Copper added, 1.57 ppm 1.61 1.21 Copper added, 3.1 3 ppm 3.16 1.22 UMean of 10 replicate determinations; std dev
Found for original sample, p p m Cu
Fe
0 1.210 0 1.23 0 1.22 0 1.20 0 1.17 0 1.22 0 1.20 0 1.20 0 1.21 0 1.22 = 0.02 ppm.
results. Means as well as standard deviations are in excellent agreement. As expected the direct measurement method (listed last) gave high results for iron, because any absorbance contribution a t 563 nm due to copper was ignored. The third listed method, involving sodium cyanide (71, is free of interference from copper. It may be employed to determine both copper and iron and is methodologically a simpler procedure than the conventional simultaneous method (listed first). Analysis of Potable Water. Simultaneous determination of iron and copper in tap water, with and without standard additions of copper and iron, yielded the results summarized in Table VII. Recoveries of added copper and iron proved quantitative. The relative standard deviation of 1.6%for the iron results compares favorably with customary levels of spectrophotometric precision. No copper was found in the unspiked samples by the method employed. Absence of any appreciable concentration of copper was confirmed qualitatively by a separate test using an extractive procedure with PDT(7). Samples of tap water, some spiked with known amounts of copper, were analyzed by the alternate method involving sodium cyanide described in Experimental. In each case, the concentration of iron found was between 1.20 and 1.22 ppm, in excellent agreement with the results in Table VII. Concentrations of added copper found for four different spiked solutions (with the difference between added and found ppm in parentheses) are as follows: 0.38 (+0.07),0.62 (-O.Ol), 1.61 (+0.04), and 3.16 (+0.03) ppm. Thus the alternate method yielded results as reliable and precise as those obtained by the conventional simultaneous determination method. Determination of I r o n in Beer. As a test of its versatility, 2,4-BDTPS was employed for the selective determination of iron in several different beers after wet oxidation with perchloric and nitric acid. The high concentration of perchlorate ions caused no difficulty since the iron(I1) chelate remained soluble, unlike that of typical unsulfonated ferroin-type ligands. The results, compiled in Table VIII, reveal that iron recovery is quantitative from spiked samples; hence, presumably also from unspiked samples. Added amounts of copper are without adverse effect. The three estimates of standard deviation obtained for the method applied to the three samples agree within expected error. Relative precision, which suffers as a result of the relative low iron concentrations of the samples, could be improved by taking larger samples for wet ashing. Although highly sensitive for iron and free of interference
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ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
Sample
cu
Fe
Total
Original
Domestic beer, aluminum can
0 0
0 0 0.276 0.276 0.27 6 0.276
0.046 0.045 0.314 0.317 0.314 0.331
0.046 0.045 0.038 0.041 0.038 -0.055 X = 0.044 S = 0.006
0 0 0.276 0.276 0.276 0.276
0.132 0.127 0.406 0.405 0.406 0.394
0.132 0.127 0.130 0.129 0.130 -0.118 X = 0.128 S = 0.005
0 0 0.276 0.276 0.276 0.276
0.08 3 0.080 0.354 0.368 0.356 0.374
0.083 0.080 0.078 0.092 0.080 -0.098 X = 0.085 s = 0.008
0 0 0.784 0.784
Domestic beer, glass bottle
0 0 0 0 0.7 84 0.784
Imported beer, glass bottle
0 0
0 0 0.7 84 0.784
from copper, the selective determination method requires a 15-minute heating period for complete color development. If numerous samples are to be routinely analyzed it is recommended that one of the two simultaneous methods, described above for water or blood serum, be used. For essentially the same amount of effort and operator time, both copper and iron can thus be determined. LITERATURE C I T E D H. Diehi, G. F. Smith, L. Mc Bride, and R. Cryberg, "The Iron Reagents: Bathophenanthroline, Bathophenanthroline Disulfonic Acid, 2,4,6-Tripyridyl-s-Triazine, and Phenyl-2-Pyridyl Ketoxime", 2nd ed., G. Frederick Smith Chemical Co., Columbus, Ohio 1965. H.Diehl, G. F. Smith, A. A. Schilt, and L. Mc Bride, "The Copper Reagents: Cuproine, Neocuproine, Bathocuproine", 2nd ed., G. Frederick Smith Chemical Co., Columbus, Ohio, 1972. F. H. Case, "A Review of Syntheses of Organic Compounds Containing the Ferroin Group", G. Frederick Smith Chemical Co., Columbus, Ohio, 1960. A. A. Schilt, "Analytical Applications of 1,lO-Phenanthroline and Related Compounds", Pergamon PTess, New York, N.Y., 1969. A. A. Schilt and G. F. Smith, Anal. Cbim. Acta, 15, 567 (1956). A. A. Schilt and W. C. Hoyle, Anal. Cbem., 39, 114 (1967). A. A. Schilt and P. J. Taylor, Anal. Cbem., 42, 220 (1970). P. Trinder, J. Clin. Patbol.. 9, 170 (1956). B. Zak, Clin. Cbim. Acta, 3, 328 (1958). D. Blair and H. Diehl, Talanta, 7, 163 (1961). L. L. Stookey, Anal. Cbem., 42, 779 (1970). 6.Zak, E. Epstein, and E. S.Baginski, Microcbem. J., 14, 155 (1969). E. Zak, E. S. Baginski, E.Epstein, and L. Weimer, Clin. Cbim. Acta, 29, 77 (1970). P. Carter, Anal. Biocbem., 40, 450 (1971). F. H. Case, J. Heterocycl. Cbem., 7, 1001 (1970). A. A. Schilt, C. D. Chriswell, and T. A. Fang, Talanta, 21, 831 (1974). B. M. Cuibertson and G. R. Parr, J. Heterocycl. Cbem., 4,422 (1967). G. W. Ewing, "Instrumental Methods of Chemical Analysis", 4th ed., McGraw-Hill, New York, N.Y., 1975, Chap. 3. A. S.Levens, "Nomography", J. Wiley, New York, N.Y., 1946, pp 25-36. R. L. Cryberg and H. Diehi, Proc. lowa Acad. Sci., 70, 184 (1963). R. M. Lincoln, A. S.Carney, and E. C. Wagner, lnd. Eng. Cbem., Anal. Ed., 13, 358 (1941). C. R. Noiler, "Chemistry of Organic Compounds", 3rd ed., W. B. Saunders, Philadelphia, Pa., 1965.
RECEIVEDfor review January 23,1976. Accepted March 11, 1976.
