Documentos de Académico
Documentos de Profesional
Documentos de Cultura
CHIMICA
ACTA
Analytica
School of Applied Biological and Chemical Sciences, Vnioersity of Ulster, Coleraine, Co. Deny BT52 ISA, UK
1995; accepted
18 December
1995
Abstract
The model ligand I-(2-pyridylazo)-2-naphthol
(PAN) has two pK, values of 2.5 and 11.2 corresponding
to the
pyridinium ion, and the phenolic group, respectively. The related chelating agent, 2-(S-bromo-2-pyridylazo)-5-diethylaminophenol (PADAP), has pK, values of 1.O, 3.0 and 11.2 corresponding to the 3-bromopyridinium
ion, the NJ-diethylanilinium ion and the phenolic group, respectively. On chelation of PADAP with Cd(II), C&I), Ni(II), Z&I) and Pb(II) a
1:l stoichiometry is found in the intermediate pH range 4-9, indicative of square planar or tetrahedral complexes. Co(I1)
forms a particularly stable chelate with a 1:2 stoichiometry over the pH range O-14 with the other chelates showing greater
lability when investigated by W-visible
spectrophotometry.
Adsorptive stripping voltammetry (AdSV) is compared to
capillary zone electrophoresis
(CZE) for the detection and determination of trace concentrations of metal ions (CdII),
Cu(II), Cd(II), Zn(II), Ni(I1) and Pb(II)) as their PADAP chelates. Limits of detection CLODS) for Cd(II), Zn(II), Pb(II) and
Co(II) were 8.3, 4.1, 3.0 and 0.5 X lo-* mol dme3, respectively, using the AdSV method with Cu(II) and Ni(I1) not giving
reproducible cathodic signals as their respective chelates. CZE was performed using 1 X 10m4 mol dme3 PADAP in the run
buffer and gave higher LODs than AdSV but better selectivity. Comparison between the two techniques is made for the
determination of Co010 in vitamin B,,. The effect of the presence of vitamins from the A, B and C groups following
destruction of the corrin ring system by UV digestion prior to chelation with PADAP was also investigated by CZE to reveal
100% signal recovery in all cases with 3% relative standard deviation following 5 consecutive 30 s hydrodynamic injections.
Keywords:
UV-Visible spectrophotometry;
(PADAP); Cobalt; Vitamin B,,
2-(5-Bromo-2-pyridyiazo)-5-diethykrninopheno~
1. Introduction
* Corresponding author.
0003-2670/%/%15:00
0 1996 Elsevier Science B.V. All rights reserved
SSDI 0003-2670(95)00624-9
The azo dyes comprise the largest group of organic reagents used in spectrophotometric
analysis
98
and include such reagents as PAN, PAR and Arsenazo III. 2-(S-Bromo-2-pyridylazo)5-diethylaminophenol (PADAP) is a derivative of PAN and
PAR and forms the basis of highly sensitive methods
for trace metal determination, with a molar absorptivity (E) often > 1 X lo5 1 mol- cm- [l]. In
recent years PADAP has been used as a relatively
non-selective means of chelating metal ions prior to
their determination by adsorptive stripping voltammetry (AdSV), e.g., bismuth 121, chromium(II1) 131,
titanium(IV) [4] copper(B) [5], iron(II1) [6] and vanadium(V) [7] have been determined this way. Individual metal determinations were reported down to limits of detection of 1 X lo-* mol dme3 (for Cu(I1)).
The use of capillary zone electrophoresis (CZE) for
the selective detection and determination of trace
concentrations of metal ions following chelation with
a suitable chromophore is gaining momentum as
evidenced by the growing number of publications in
this area. For example Motomizu et al. {8] have used
the chelating agent 5-Br-PAPS and Iki et al. [9] and
Regan et al. [lo] the agent 4(2-pyridylazo)resorcinol
(PAR) for this purpose, achieving good separations
with limits of detection in the range 1 X 10p6-1 X
10m8 mol dme3. Swaile and Sepaniak [II] used
5-sulpho-8-quinolinol along with laser excited fluoresence to detect Ca(II), Mg(I1) and Zn(I1). The three
metal ions could be detected at ng cmF3 levels and
in complex matrices such as blood serum. The present paper is concerned with (a) a W-visible spectrophotometric study of the acid-base behaviour of
PADAP and the related model ligand I-(2-pyridylazo)-2-naphthol (PAN) in order to establish the
charge states of PADAP at particular pH values; (b)
a W-visible spectrophotometric study of the chelation of PADAP with Co(B), Cu(II), Ni(I1) and Pb(I1)
over the pH range O-14 to study the extent of the
reaction of these metal ions with PADAP and to
select optimum wavelengths for chelate detection in
CZE; (c) a study by AdSV and CZE of the chelates
of PADAP with CdII), Cd(I1) Cu(II), Ni(II), Zn(I1)
and Pb(I1) and their analytical applications and (d)
application of the AdSV and CZE study to the
detection and determination of Cd110in vitamin B ,2
(cyanocobalamin) following destruction of the corrin
ring system by a W digestion procedure and chelation of the freed cobalt with PADAP. The effect of
other vitamins of the A, B and C groups, as would
2. Experimental
2.1. Apparatus
The AdSV studies of the metal chelates were
carried out using a Metrohm 646 VA Processor, a
Metrohm 647 stand with the multimode electrode
(dropping mercury electrode (DME) and hanging
mercury drop (HMDE)) and the 675 VA sample
changer, controlled by the 646 VA processor
(Metrohm, Herisau, Switzerland), which can automatically and sequentially process up to 10 samples.
A three-electrode system was used throughout consisting of a Ag/AgCl reference electrode, the HMDE
as the working electrode and a Pt counter electrode.
Linear regression analysis was applied to peak height
values.
Electrophoretic separation of the metal chelates
was performed using a SpectraPhoresis 1000 instrument (Thermo Separation Products, Stone, UK) fitted with a untreated fused silica capillary, 75 cm x 50
pm, (Composite Metal Services, Hallow, UK) with a
detector window burned at 68 cm. All instrument
control and data handling were performed using
SpectraPhoresis software. Metal chelates were introduced into the capillary by hydrodynamic injection
and monitored using a W-visible diode array detector (DAD) in the visible region, 550-585 nm. Peak
areas were calculated by integration.
UV digestion was carried out using a Metrohm
UV Digester 705. The pH was measured using a
AGB Model 3050 pH meter (AGB, Carrickfergus,
UK).
The spectrophotometric study of the ligands and
the metal chelates was performed using a HewlettPackard 8451 diode array spectrophotometer (Hewlett
Packard, Palo Alto, CA).
99
2.3. Procedures
loo
DA.
Oxspring et al./Analytica
tl.0
k (nm)
Fig. 1. UV-visible
spectrophotometric
behaviour of 0.2X 10m4
mol dme3 (I) in the pH regions O-5 and 10-13. The latter study
has its absorbance values moved up 0.45 for all spectra.
1.4.
1.2 -
1.0 -
0.6 -
0.6 -
Fig. 2. UV-visible
spectrophotometric
behaviour
HA and A-,
101
0.8
1
s!
s
0.6
0.4
~0!1-1-11.1-11.1.1.11.1-1(
0
1 2
9 IO 11 12 13 14
PH
Fig. 3. Variation of absorbancewith pH for 0.2X 10e4 mol dme3
PADAP at 448 nm, 510 nm and 534 mn. (0) 448 nm; (A) 510
nm; (0) 534 nm.
agreement with the predicted pK, value as calculated from the equation pK, = 5.06-3.46&r [12]. In
this case a,,,, for an OH group is 0.13 and opara for
an aniline substituted by the group -N=N-C,H,
is
0.57. Hence the predicted pK, value is 5.06-3.46
(0.57 + 0.13) = 2.46. The pK, of 11.2 again corresponds to the phenolic group.
The W-visible spectra again illustrate the existence of tautomeric equilibria with the two major
absorptions at ca 450 nm and ca. 520 nm presumably
representing azo and hydrazone tautomers, respectively. It would appear from the spectra that when
the 3-bromopyridine group is protonated at pH 0 the
molecule exists exclusively in the azo form with
only the 450 nm absorption band present. At pH 13,
when the phenolic group is ionised and the 6-membered ring structure is broken down, the 520 nm
absorption band representing the hydrazone is predominant. The W-visible spectra for PADAP (III)
are further complicated by tautomeric equilibria involving the NJ-diethylamino
group where imine
tautomers such as (IV) have been proposed [ 131.
102
OP .
0
metakligand ratio
Fig. 4. Variation of absorbance of Co(H), Cu(Il), Ni(I1) and Pb(II)
chelates of PADAP, measured at their respective A,,, vatues,
with varying metal/PADAP
ratio in the range 0.1: 1 to 41. (0)
Lead; (A ) nickel; ( X ) cobalt; ( q ) copper.
103
I
58.20
$
B
6.39
::
9
4.96
-0.oco61
0.06
2.00
4.00
&
6.00
6.00
lO.c!O
Time (min)
Fig. 6. CZE of a equimolar mixture of Cd(I1) (4.98 min), CdII) (5.20 min), Cu(I1) (6.04 min), Zn(I1) (6.39 min), Ni(I1) (6.72 min) and
Pb(I1) no signal (ah at 1.6 X 10-s mol dmm3) in a run buffer of 0.05 mo1 dmm3 sodium acetate with 20% acetonitrile and IO- 4 mol dm-3
PADAP, pH altered to 6.0 with 1 mol dme3 orthophosphoric
acid.
104
Table 1
CZE conditions for detection and determination of selected metal ions using PADAP: efficiency values, LOD values, calibration ranges and
correlation coefficients
Metal ion
Detection
wavelength (nm)
PI-I
Efficiency: number
of plates(N)
LOD
(mol dmm3)
Correlation
coefficient (n = 4)
cd10
587
548
554
556
576
548
7.0
6.0
7.0
7.7
1.7
6.0
1.6 X 10
6.6 x lo4
1 x106
2.8 X lo*
8.6 x 103
2.5 X 10
5x
5x
5x
1x
1x
1x
5x
5x
5x
1x
1x
1x
0.9982
0.9905
0.9910
0.9988
0.9958
0.9991
Cd(R)
C&II)
Ni(I1)
Pb(II)
Z&I)
area of the Pb(I1) chelate decreases with pH decrease. The efficiency in terms of number of plates
N where N = 16 (t,/W>* was calculated for the
individual metal chelates (Table 11, which showed
that the Co, Cd, Cu and Zn chelates have high
efficiencies in the 6.6 x IO4 to 1 X lo6 range. The
Pb and Ni chelates have relatively poor efficiency
values of 8.6 X lo3 and 2.8 X lo* respectively due
to their poor peak shape.
The elution order of the six metal ions does not
agree with the charge/mass ratio for each of the
chelates. Fig. 6 shows the elution order of the metal
chelates at equimolar concentrations (1.6 X 10m5 mol
dme3). Cd(I1) elutes first - which is surprising due
to its relatively high atomic mass (112.41) and the
comparatively low charge/mass ratio ( z/m) of 4.33
x 10e3 for the chelate (allowing for a 1:l stoichiometry). The order Co(B), Cu(II), Zn(I1) and
Ni(I1) is in agreement with work by Iki et al. [9] and
Regan et al. [lo] using the PAR chelate. On an
atomic weight basis, it would be expected that the
Ni(I1) chelate would elute before that of Cu(I1) but
the reverse is observed. Ni(I1) elutes later which
could be expected because of its poor peak shape
which is an indication that the Ni(I1) chelate is
interacting with the capillary wall. The Pb(I1) chelate,
with a significantly lower z/m value than those of
Cu(I1) and Ni(II), elutes last at those pH values
where it is detected (e.g., pH 7.7).
The limits of detection (LODs) for the six metal
ions were estimated
at the pH at which each chelate
gave its most intense signal i.e., greatest peak height.
Pb(I1) and Ni(I1) gave their most intense signals at
higher pH values (e.g., pH 7.71, even though the
signals were somewhat broad. Cd(I1) and Zn(I1) are
10-7
10-6
10-e
10-6
10-6
10-s
10-7
10-6
10-6
10-6
10-6
10-6
Co(I1) will complex with PADAP to form a particularly stable chelate which can be detected and
determined selectively by AdSV and CE. Both techniques were compared for their ability to determine
Co(II1) in vitamin B,, (cyanocobalamin) following
destruction of the corrin ring system by UV digestion using H,O,.
Table 2
AdSV conditions for detection and determination of selected
metal ions using PADAP, LOD values and correlation coefficients
Metal
ion
LOD (X lo-*
Reduction peak
potential of PADAP mol dm-)
chelate (mVI
cd10
-670
- 620
- 1.071
-580
Cu(II)
Cd(R)
Zn01)
Ni(II)
PdII)
Correlation
coefficient
from logtpeak
ht.) - logkonc.)
plots (n = 6)
0.5
0.9908
8.3
4.1
0.9944
0.9999
3.0
0.9954
Using the methodology outlined in the Experimental section, AdSV of the UV digested
cyanocobalamin was performed giving an LOD for
the free cobalt of 3.5 X 10m9 mol dmm3 and a
correlation coefficient of 0.9936 (n = 6) for a calibration plot in the range 1.4 X 10--4X lo-* mol
dm-. Using CZE a limit of detection of 5 X lo-
mol dmM3 was achieved, showing that all the cobalt
.
.
.
m vnamm B,, had been released by the UV digestion procedure and had been subsequently chelated
with PADAP. Linear regression analysis on the calibration plot over the concentration range 1 X 10e45 X 10d7 mol dmd3 gave a correlation coefficient of
0.9999 (n = 4).
The recovery of this CZE cobalt signal from
vitamin B ,2 was investigated in the presence of other
vitamins that occur in multivitamin mixes, namely,
retinol (A), thiamine hydrochloride (B ,), riboflavin
(B,), niacinamide (B,), pyridoxine (B,) and ascorbic
acid (0. At a Co(B) concentration of 2.5 X 10e5
mol drnm3 in the presence of equimolar concentrations of the above mentioned vitamins, following UV
digestion of the mixture the CoUII) chelate gave a
migration time of 4.52 min with 100% signal recovery. Furthermore 100% signal recovery was again
achieved for 1.25 X 10m6 mol dmW3 Co(II1) from
vitamin B ,z in the presence of 1.4 X 10e4 mol
drnd3 concentrations for each of riboflavin, thiamine
hydrochloride, ascorbic acid and retinol, 1 X 10m2
mol dmm3 nicotinamide and 1.4 X 10e2 mol dme3
pyridoxine. The relative standard deviation of five
consecutive
30 s hydrodynamic injections of a 5 X
10e6 mol dmm3 solution was calculated as 3%. This
CZE method would therefore appear to have the
potential to measure trace concentrations of cobalt in
biological samples once binding of the cobalt to
biomolecules and other interferences are destroyed
by the UV digestion procedure.
4. Conclusions
The pK, values of PADAP were determined to
be 1.0, 3.0 and 11.2, giving an indication of the
105
charge states of PADAP at various pH values. Determination of Co(B), Cd(B), Z&I) and Pb(I1) using
PADAP can be performed by AdSV which gives
individual metal chelate determinations with LODs
in the nanomolar range. AdSV offers particular good
selectivity and sensitivity for the determination of
Co(B) in a Zn(II), Cd(B) and Pb(I1) mixture with a
LOD of 5.0 X 10e9 mol dmm3. CZE of the overall
positively charged chelates in comparison gives
higher LODs for the metal ions, with the Co(B)
chelate giving the lowest LOD of 5 X lo- mol
dmm3. Both AdSV and CZE show good sensitivity
and selectivity for the determination of Co(II1) in a
UV digested cyanocobalamin sample with LODs
comparable to those obtained for free Co(B). The
recovery of the CZE cobalt signal from vitamin B,,
is found to be unaffected by the presence of equimolar and higher concentrations of other vitamins found
in multivitamin mixtures following UV digestion.
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