FLUORIMETRIC ANALYSIS FOR MICROQUANTITY COPPER
DETERMINATION
K. Yu. Shunyaev, N.V. Pechishcheva, A.A. Shchepetkin
Institute of Metallurgy of UB RAS,
101, Amundsen st., Ekaterinburg, Russia
shun@ural.ru
It is necessary to determine microquantity of copper in great
numbers of materials, including environment objects, food and
metallurgical products.
Copper is a nutrient required for many biochemical and
physiological function, but high concentration of this metal may damage
human health and many organisms such as certain algae, fungi and many
bacteria and viruses [1-5]. A lot of European and Russian directives
establish permissible concentration of copper in various substances, for
instance in drinking and waste water, air, soil, alloys and ceramics for
food preparation, textile [6-9].
Furthermore, determination of small amount (0.0001-0.010 % wt
level) of copper is requirements for many metallurgical products such as
pure metals (Al, Mg, Cd, Sn, Pb, Au, Ag), nickel alloys, titanic alloys,
semi-conductors, etc.
Many new and modified techniques can be used for low level
copper quantification, among them atomic absorption spectroscopy,
inductively coupled plasma atomic emission spectroscopy,
electroanalytical chemistry, electrospray ionization mass spectroscopy
[4, 5, 10].
While these methods have some pros and cons, fluorescence
techniques has merit in a sense that it is sensitive, convenient and
simpler than other methods, not requiring expensive analytical
instrumentation. Among the molecular spectroscopy methods,
fluorescence has the lowest detection limit. One can found the review of
fluorimetric techniques of the copper determination with several organic
reagents in Table 1 [11] and in the paper [12].
Usually, two versions of fluorimetric techniques are used in
chemical analyses of copper-content objects.
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Table 1. Comparison of the main characteristics for fluorimetric technique
determination of copper with several organic reagents [11]
370/440
Linear range
(mg/l)
0–2
РН=8, heated, extracted
Neocuproine
Vc-OPDA
560/570
350/420
0,001-0,006
0-0,008
РН=9, exracted
РН=6.9
3PTNCAP
308/403
0,0004-0,064
РН=5.6, boiled for 30 min
3PRCAP
SDBH
305/405
300/410
0-0,048
0-0,080
РН=5.6, boiled for 25 min
РН=9.0, boiled for 45 min
Тiamine
370/440
0,00024-0,4
рН=12, stood for 30 min
FСPAQ
328/368
0,004-0,14
FCPBSQ
326/362
0,001-0,2
BAQABP
296/382
0,003-0,15
рН=5.4, boiled for15 min
Tween-80 as surfactant
рН=6.4, boiled for 5 min
Tween-80 as surfactant
рН=8.4, boiled for 20 min
Tween-80 as surfactant
Reagent*
ex/fl
BSTMED
Experimental condition
Interfering
ions**
Fe3+, Co2+,
Ni2+
No
Hg(II),
Cr(VI),
Sn(IV), V(V)
Pb2+, Fe3+,
Bi3+, Ag+
Pb2+, Fe3+
Pb2+, Fe3+,
Al3+, Co2+,
Ni2+
Fe3+, Co2+,
Mn2+, Fe2+
No
No
No
* The
abbreviation of the reagents represented as follows:
BSTMED: bis-(salicyadehyde)tetramethyl-ethylenediimine;
Vc-OPDA: ascorbic acid and o-phenylenediamine;
3PTNCAP: 3-tolyl-5-(4-nitro-2-carboxylphenylazo)-2-thioxo-4-hiazolidone;
3PRCAP: 3-phenyl-5-(2%-carboxylphenylazo)-2-thioxo-4-hiazolinone;
SDBH: salicyladehydebenazalhydrazone;
FСPAQ: 5-(3-fluo-4-chlorophenylazo)-8-aminoquinoline;
FCPBSQ: 5-(3-fluo-4-chlorophenyl-azo)-8-benzenesulfonamidoquinoline;
BAQABP: 4,4-bis(8-aminoquinoline-5-azo) biphenyl
** Could produce interference at the same concentration of copper(II).
Quenching of the fluorescence as a basis for copper
quantification
The largest group of the methods of the copper determination is
fluorescence quenching techniques. In most cases, complexation of
copper ions with fluorescent ligands results in strong interactions
between both of them. This interaction, in particular intramolecular
charge transfer, magnetic interactions usually cause a strong or even
complete quenching of the fluorescence (it is axiomatic that
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paramagnetic species quench the fluorescence). Most of the ligands
described for this kind fluorescent techniques, can be classified as
broadband complexing agents. Hence, the presence of ubiquitos metal
ions can interfere with the determination of copper.
One can found the examples in the papers [13-16]. Copper have
been determined in diluted brass samples and the stream water samples
in the concentration range 0.03 – 0.64 mg/l with 4,5-dihydroxy-1,3benzenedisulfonic acid (Tiron) [13] and of the boiler water samples in
the range 0.0005-0.025 mg/l with [N,N-di(carboxymethylaminomethyl]fluorescein (fluorexon) [14].
In [15] sensitive method for copper determination in proteins,
based on quenching of bathocuproine disulfonate fluorescence by
cuprous copper at neutral pH in the range of 0.02 – 0.13 mg/l Cu, other
metal ions (iron, manganese, zinc, cadmium, cobalt, nickel) do not
interfere when assayed at concentration 10-fold higher than that of the
copper.
In [16] an optical sensing scheme for determination of copper in
drinking or waste water based on static quenching of the fluorescence of
Lucifer Yellow immobilized on anion exchanger particles, embedded in
a hydrogel, have been described. Linearity range is 0.00063-6.3 mg/l of
Cu. Heavy metal ions such as cadmium, zinc, lead, iron, cobalt, nickel
are not influencing the fluorescence signal intensity.
There are many report of chemosensor for Cu(II) (e.g., works of
Fabrizzi et all. [17-20]) where the sensing action involves fluorescence
quenching. In general, the sensors have been designed following a twocomponent approach, i.e. by linking a selective ligand for Cu(II) to a
light-emitting component (usually anthracenyl moiety), that may be
quenched by a variety of mechanisms including photoinduced electron
transfer (PET) from the fluorophore to the metal center, involving the
Cu(II)/Cu(I) couple, and electronic energy transfer (EET).
Some of the methods of the copper determination are based on the
quenching of rare earth [21] and theirs chelates [22] fluorescence. The
method presented in [22] provides a simple specific determination of
copper at 0.05-3 mg/l range. Time-resolved fluorimetry have been
applied, that has advantages over conventional technique due to
possibility of its application to inhomogeneous, turbid, highly lightabsorbing samples, Interference by static quenching of other heavy
metals (Cd(II), Hg(II), Zn(II), Pb(II) ions) is efficiently discriminated,
175
but dynamic quenchers (Cr(III), Mn (II), Ni(II), Co(II), Fe(III) ions)
interfere copper determination.
Enhancing of fluorescence as a basis for copper determination
In in rare instances on can observe the enhancing of fluorescence
during complexation of the fluorophore with copper ions. Compounds,
which exhibit an increase in fluorescence intensity (chelation enhanced
fluorescence, CHEF) in the presence of the ion, are very attractive due to
the greater sensitivity of metal determination.
In the paper [11] three aqueous spectrofluorimetric methods for
the determination of copper based on the reaction of three new reagents
– 8-aminoquinoline-5-azo derivatives – with copper in the present of the
surfactant are proposed. They are used to determine copper in ore, alloy,
hair and water samples with satisfactory results in the range of Cu
concentration 0.001 – 0.2 mg/l.
Authors of the studies [23, 24] were making a supposition about
cause of fluorescent reviving of some chemosensors. In most of the
reported Cu(II) sensors the fluorescent moiety is placed far away from
the chelating moiety and the linker heteroatom of fluorophore does not
participate in complexation. As a result electron transfer from
heteroatom to fluorofore causes fluorescence quenching (Fig. 1A). It has
been supposed, that if the linker heteroatom of the fluorophore
efficiently participates in complexation with Cu(II), it must suppress the
process of PET from the heteroatom (generally amine nitrogen) to the
fluorophore (Fig. 1B). In the case of this effect overweighing the
contrary effects of electron transfer quenching by paramagnetic Cu(II),
net fluorescence enhancement would be observed.
Fig. 1. The different mechanisms of sensing action [23].
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A two thioether and three amine units based CHEF which detects
0.06-0.13 mg/l of Cu(II) in CH3CN:H2O (4:1) are reported in [22] and
two 4-(aminoalkyl)aminonaphthalimide compounds which exhibit
fluorescence enhancement in the presence of Cu(II) (in the range 0.3-1.0
mg/l), Mn(II) and Ni(II) (in the range 10-100 mg/l) in the 2-propanol
medium are reported in [24].
Chemiluminescent reaction for copper determination
A number of investigations regarding the use of
chemiluminescence (CL) have been proposed for copper quantification.
This kind of emission is not requested the external excitation source and
generated by release of energy from a chemical reaction. Copper (II)
often catalyze the reaction of organic compounds such as luminol,
lecigenin, lophine with H2O2, usually in alkaline medium. Formation and
following reorganization of organic compound with the relative weak OO bond liberates a large amount of energy as luminescence. Emission
intensity is dependent upon the concentration of copper (II). Such
methods are usually very sensitive, have low limit of determination, but
are not selective enough (they are affected by other transition metals
ions).
The determination of copper in zinc and pure alkali with luminol
+ H2O2 + Cu (II) is described in [25]. The authors of [26] was
determining of Cu(II) contents in immunoglobuline, foods and medicine
with system luminol + H2O2 + copper amino acid chelate in
concentration range 0.001-0.1 mg/l of Cu (II). Fe(III), Fe (II), Cr (III)
interfere the analysis results when exist at greater then 10-fold
ration(w/w) to Cu(II).
Chemiluminescent system fluorescein – NH2OH- OH have been
developed for the determination of Cu (II) in serum [27] (range of
linearity of calibration graph is 0.001-0.02 mg/l). The system is selective
for copper except in the presence of Fe (II), Fe (III), and Co (II). The
following mechanism of CL processes is proposed: first an autoxidation
reaction to give hydroxylimine took place in the basic aqueous solution
with copper(II) ions as catalyst. The emission (max = 478.6 nm) was
thought to be from the exited molecular oxygen species (excimers)
which were produced from hydroxylamines reacting with dissolved
oxygen, catalyzed by copper(II). The excited molecular oxygen species
177
then transferred energy to the fluorescein (FL) molecule, forming exited
organic molecules, and the emission (max = 560 nm) was generated:
(O2*)2 + FL  2O2 + FL*
FL*  FL + h.
Commonly used and standardized in Russia fluorimetric method
of copper determination in the samples of air and surface water with
lumocupferron (L) is also basing on the chemiluminescence [28].
Lumocupferron is not fluorescing in water solution, but at addition of
Cu(II) ions in slightly alkaline medium green emission is observed. The
reaction pass the step of the emitting lumocupferron-dimer
formation [29]:
Cu2+ + L-  CuL+
CuL+ + L-  CuL2
CuL2  Cu2+ + L22L22-  2L- + h.
It is necessary to note, that other important variety of copper
quantification techniques basing on luminescence phenomena exist. For
instance, a series of investigation deals with phosphorescent copper
determination [29-31], including low-temperature techniques [29, 30],
the methods, using sorption phenomenon [30]. The emission observed
has d*d transition of Cu(I) origin. This group of method is very
sensitive and selective, but some complicated for practical usage.
Conclusion
There is a great number of luminescent technique of copper
quantification, but all of them have specific area of application,
restricted by their range of linearity, availability of equipment and cost
of materials, complication of ligand synthesis. Lot of them are extraction
fluorimetric methods and imply handling of toxic organic solvents, the
directly aqueous fluorimetric methods are scarce. Sometimes
supplementary operations are obligatory, for instance extraction,
sorption, boiling, surfactant and masking regents addition.
It is necessary to choose the most convenient method, taking into
account also possible interference species, rapidity and simplicity.
178
Therefore developing of new fluorescent materials and techniques
both selective and sensitive toward the copper ion remains the task of
current importance.
This work was supported by RFBR grant № 06-03-32981 and
grant “Leading scientific schools” NSh-5468.2006.3.
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