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DOUGLAS
J. HENNESSY
A N D GARYR. REID
Dingnostic Chetnicn1.s Lilnited. Chnrlotteto~t~n.
P.E.I.. Cr~nnrlrrC I A 4H5
AND
FRANK
E. SMITH'A N D STEPHEN
L. THOMPSON
Chetnistry Departtnent, Unii~er.sir~
oj' Pri11c.c. Edivtrrrl 1.slnncl. Chrrrlotfrto~t~n.
P.E.I.. Ccrnnrlr C I A 4P3
DOUGLAS
J. HENNESSY.
GARYR. REID, FRANK
E. SMITH.
and STEPHEN
L. THOMPSON.
Can. J. Chem. 62, 721 (1984).
Ferene iron reagent, 3-(2-pyridyl)-5,6-bis(2-(5-furylsulfonic acid)-l,2,4-triazinc. disodium salt, n~onohydrate,has bccn
synthesized and characterized. Results of a study of its complex formation with iron(ll) including A,,,.,,, E,,,.,,, and log K values
are reported.
DOUGLAS
J . HENNESSY,
GARYR. REID, FRANK
E. SMITH
ct STEPHEN
L. THOMPSON.
Can. J. Chem. 62, 721 (1984).
On a synthCtisC et caractCrisi le ferenc: Ic sel de (pyridyl-2)-3-bis-5.6-(furyl-2-acide
sulfonique-5)-triazine-1,2,4-disodium
monohydrati. On rapporte les rksultats de I'Ctudc dc la formation d'un complcxc avec le fcr(ll), parmi lesqucls on retrouvc
Ics valcurs de A ,,,;,,, E,,,,, et log K.
[Traduit par Ic journal]
Introduction
Bidentate chelating agents containing the a-diimine or ferroin group have been used for many years as chromogenic reagents for the determination of iron and copper ( I -5). A major
step forward in the development of reagents of this type came
with a discovery by Case and Smith and their respective coworkers (6-8). They found that the incorporation of phenyl
groups in positions para to the ferroin group led to greatly
enhanced absorption by the metal chelates, resulting in considerable improvements in the sensitivity of the resulting reagents.
~ollowing this discovery, the widely used reagents bathophenanthroline (6,7), bathocuproine (9), terrosite (I O), PPDT
(1 I), and PDT (12) were introduced. These phenyl-substituted
compounds and their metal chelates all suffer from the disadvantage of having very low solubilities in water. This makes
them unsuitable for use in the rapid, highly automated procedures necessary for dealing with large numbers of samples.
The sulfonation of bathophenanthroline by Trinder (13) led
to a water soluble derivative which retained the favourable
chromogenic characteristics of the parent compound even after
sulfonation. Subsequently, the sulfonated, water-soluble derivatives of bathocuproine (14), PDT (15), and terrosite (16, 17)
have all been reported, as well as those of a series of new
phenyl-substituted triazines recently synthesized by Case (18,
19). The chromophoric enhancement observed in the phenylsubstituted reagents is presumably a result of the phenyl groups
acting in such a way as to favor the charge transfer process
which causes the intense colors of the iron(l1) and copper(1)
complexes. This being the case, it was considered that the
introduction of furyl groups into the chelating molecules might
well lead to a similar chromophoric enhancement. The compound 3-(2-pyridy1)-5,6-bis(2-fury1)-l,2,4-triazine containing
two furyl groups was therefore synthesized. Sulfonation of this
triazine has led to the isolation of a new, highly sensitive and
water-soluble colorimetric reagent for iron, the disodium salt of
3-(2-pyridy 1)-5,6-bis(2-(5-fury1 sulfonic acid))- 1,2,4-triazine,
hereafter referred to as Ferene (Fig. 1).
S,03Na
Experimental
Synthesis of 3-(2-pyrirl~~l)-5.6-bis(27f~r~~l)-1,2,4-triazit~e
To a stirred suspension of 16 g (0.84 mol) of furyl in 130 mL of
isopropyl alcohol heated to 60C. 13 g (0.96 mol) of picolinamidrazone (20) (callcd 2-pyridyl hydrazidine in the reference) was
added portion-wise over a period of 5 min. The stirring at 60C was
continued for 112 h. The solid product obtained by filtration and
washing with water was suspended in 140 mL of 0.06 N hydrochloric
acid. After stirring for 2f h thc sluny was filtered. The product was
washed with water, then with hexane, and was air dried. Melting
point, 155- 157C. Anal. calcd. for CIGHIONaOZ:C 66.21, H 3.47, N
19.31; found: C 66.16, H 3.45. N 19.24.
Synthesis of the tnor~osodi~rtn
salt of 3-(2-p~~ricIyl)-5,6-bis(2-(5~ur~l
srrlfonic acid)- 1,2,4-triazine
To 22 mL of 30% fuming sulfuric acid heated to 60C, 16.5 g (0.57
mol) of the triazine was added portion-wisc with stirring so that the
temperature remained at 65 1 3C. The mixture was then warmed to
75C for 314 h after which it was poured cautiously into 44 mL of
stirred water. With the resulting solution at 60C, 90 mL of 25%
aqueous sodium chloride at the same tcinperature was added with
stirring. After standing overnight under refrigeration, the solid product
was collected by filtration, then washed with 3 X 35 mL of 10%
aqueous sodium. chloride and 2 x 35 mL of 70% aqueous ethanol,
after which it was air dried.
Syntl~esisofthe disorli~rtnsalt of 3-(2-pyricl~~l)-5,6-bis(2-(5-fL11yl
srrlfonic acid)- 1,2,4-rriazine (Ferene)
The air-dried monosodium salt was suspended in 13 mL of water
722
WAVELENGTH (nm)
FIG. 2. Visible absorption spectrum of ferrous complex of Ferene.
Determination of log K (ie. log P,) values were made as described
by Gibbs (21).
Continuous variations study
A continuous variations series with a total concentration of 1.79 x
M was performed. Ferene and iron solutions of equal concenM ) were combined such that the total volume was
tration (1.79 x
always I00 mL, i.e., 90 mL Ferene solution and 10 mL iron solution;
80 mL Ferene solution and 20 mL iron solution, etc. Again, a period
of about 5 min was allowed to ensure full color development of the
complex in each instance.
Variation of absorbance with pH
This was carried out in the manner described by Gibbs (21).
Several solutions, each having a total volume of 100 mL, were
M
made up to contain 15 mL of lo-% Ferene, 5 mL of 5 X
iron, 2 mL of I M reducing agent, and 5 mL of 2 M acetate buffer
solution. The amount of acid (I M HCI) or base (I M NaOH) added
to each flask was varied so as to give a broad range of pH values. The
remainder of each flask was filled to the mark with distilled water. A
period of 15 min was allowed for full color development of the various
solutions, at which time absorbance readings were taken on the spectrophotometer at 593 nm.
723
HENNESSY ET AL.
TABLEI. Determination of the conditional formation constant for the tris(Ferene)iron(II) complex (FeL3")
Fez+:L ratio
Conditions
I :3
[Fe"]
constant (4.48
M) ([L] varied)
[Fe"]
constant (2.24
M) ([Fez+] varied)
1.3
.'.
[F~L~'+I(I
[Fe2+]"
log,,, K"
[Ll'
3.91
10-'M
0.57
10-'M
1.71
lo-'M
1:3
1.65
lo-' M
0.59
lo-' M
1.77
lo-' M
14.7
1:3
3.42
lo-' M
0.58 x
1.74
lo-' M
15.0
3.76
lo-' M
0.72
15.1
2.12 x lo-' M
14.7
Mean value = 14.9
lo-' M
confirms that Ferene behaves as a bidentate rather than a tridentate chelating agent under these conditions.
Spectrophotometric titration experiments were carried out
with the iron concentration held at two different constant values, and also with the Ferene concentration held constant. The
results of these experiments are summarized in Figs. 4 and 5.
In each case, confirmation of the 3 : 1 Ferene: iron ratio present
in the complex was obtained. Furthermore, since excess reagent was required for complete complex formation to be
achieved, it was possible to evaluate K, the conditional formation constant, by straightforward means. The concentrations
measured and the log K values obtained are given in Table 1.
The mean log K value obtained was 14.9. This may be compared with the values reported for: bipyridine, 16.4- 17.6;
1,10-phenanthroline, 20.2-21.5, and Ferrozine, 15.6 (21,
22).
The intense blue color of the Ferene-iron(I1) complex was
found to be fully developed within 3 min at 25"C, over a pH
range of 2-7.5 in acetate buffer (Fig. 6). This is virtually the
same range as reported for formation of the Ferrozine-iron(l1)
complex in acetate buffer (21). Thus it would seem that a pH
range of 3-6 could be relied on to give the best test results.
Apart from iron, the only metal normally present in serum
which forms a coloured complex with Ferene is copper. Inter-
00
10
11
12
13
14
FINAL pH
FIG.6. Effect of pH on formation of ferrous complex of Fcrene.
ference studies with N a ' , K ' , Ca", Mg", Hg" , and Pb"
have shown that the presence of these ions, even in concentrations as high as 100 mg/L, has no effect on iron determinations using Ferene (26). Copper interference with Ferene
is similar to that reported for Ferrozine (27) and may be suppressed in a similar way. The effects of copper interference can
be almost entirely eliminated by the addition of thiourea, which
preferentially binds the copper in a copper(1) conlplex (26).
Ferene is one of the most sensitive colorimetric reagents for
iron(1l) yet reported. Both the reagent itself and its iron(l1)
complex are highly water soluble, making Ferene very suitable
for completely automated analyses on a practical scale. Two
reports on such uses have already appeared (23, 24), and there
will doubtless be many more in the future. Ferene has been
used as the basis of a kit for determination of serum iron since
1979 (25).
Acknowledgement
The authors wish to thank the Natural Sciences and Engineering Research Council of Canada for financial support.
and F. P. RICHTER.
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