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ANALYTICA

CHIMICA
ACTA
Analytica

Chimica Acta 323 (1996) 97-105

UV-visible spectrophotometric, adsorptive stripping


voltammetric and capillary electrophoretic study of
2-( S-bromo-2-pyridylazo) -5diethylaminophenol and its chelates
with selected metal ions: application to the determination of
Cd III) in vitamin B 12
D.A. Oxspring, T.J. Maxwell, W.F. Smyth

School of Applied Biological and Chemical Sciences, Vnioersity of Ulster, Coleraine, Co. Deny BT52 ISA, UK

Received 15 August 1995; revised 18 December

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,,

Stripping voltammetry; Electrophoresis;

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

DA. Oxspring er al,/Analytica Chimica Acta 323 (1996) 97-105

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

be found in a multivitamin mixture, is also evaluated.

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).

2.2. Reagents and analytes


AnalaR grade nitrate, chloride,
were used for preparing standard
metal ions (BDH, Poole, UK). The
trolytes used were all of AnalaR

and sulfate salts


solutions of the
buffers and elecgrade and pur-

DA. Oxspring et al./Analytica

chased from BDH. The ligands 2-(S-bromo-2-pyridylazo)J-diethylaminophenol


(PADAP)
and l-(2pyridylazo)-2-naphthol
(PAN) were purchased from
Aldrich (Gillingham,
UK). To ensure that PADAP
did not precipitate from its stock solution a 1 X lOa
mol dmm3 solution was prepared in acetonitrilewater (1: 1, v/v).
The vitamins
cyanocobalamin
(B ,* 1, riboflavin (B,), pyridoxine (B6), nicotinamide
(B,), thiamine hydrochloride (B ), ascorbic acid (0
and retinol (A) were also purchased from Aldrich.
All solutions were made up in Mini-Q 18 Q cm
water (Millipore).
r

Chimica Acta 323 (1996) 97-105

99

of ca. lo- mol dme3 and the above procedure was


repeated to give responses from the metal chelates.
Pb2+ was anomalous in that it did not give an AdSV
signal without the presence of trace concentrations of
Co2+. With the addition of 5 x lo-
mol dm-3

Co2+ tothe supporting electrolyte, a chelate response


was observed at -580 mV and this response increased with increasing concentrations
of Pb2+ in
the range 1 X lo--5
X lo-
mol dm-3. Repro-

ducibilities were calculated from the peak height


values for 5 consecutive determinations. The LOD
for AdSV was measured when the peak height was
three times the standard deviation of the blank value.

2.3. Procedures

2.5. CZE procedure


2.3.1. Spectrophotometric
studies of ligands and
metal chelates
A 0.5 cm3 sample of chelate solution following
its formation from 3 cm3 of 1 X 10U4 mol drne3
PADAP + 3 cm3 of 1 X 10e4 mol drne3 metal was

added to 2 cm3 of the appropriate pH buffer prior to


recording the W-visible
spectra. The pH region
O-l was covered using 1 and 0.1 mol dmp3 HNO,
and
pH
son
the

pH 13-14 by 0.1 and 1.0 mol dmb3 NaOH. The


range 2- 12 was covered by stock Britton-Robinbuffer (0.02 mol dme3) with the pH adjusted by
addition of various volumes of 1 mol dme3

NaOH. The mole ratio of each chelate was calculated


from the results of absorbance measurements made
at the wavelength of maximum absorbance of the
chelate. The PADAP concentration was kept constant at 1 X 10e4 mol dme3 and the metal varied
from a l:O. 1 ratio to a 1:4 ratio.
2.4. AdSV
For the voltammetric studies 1 X 10e3 mol dme3
PADAP stock solution was prepared in 1:1 (v/v>
ethanol-water. The supporting electrolyte of 0.1 mol
dme3 NH3/NH4N03 (20 cm3>, containing 1 X 10m6
mol dm- 3 PADAP, was deaerated for 5 min by
bubbling nitrogen. A mercury drop (0.6 mm21 was
then dialled on the HMDE and pre-electrolysis carried out at - 2UOmV for 180 s with stirring. After a
10 s settling period, the solution was cathodically
polarised at a scan rate of 6 mV s- . This gave the
voltammetric response of the ligand. Metals were
then spiked into the solution to give concentrations

Prior to use the fused silica capillary was washed


through with methanol for 5 min at 40C followed
by 0.1 mol dmw3 sodium hydroxide and Mini-Q
water for 5 min at 60C. The capillary was finally
washed through with run buffer for 5 min at 25C.
Separation was performed with a run electrolyte of
0.05 mol dmm3 sodium acetate, 20% (v/v) in acetonitrile and 1 X 10e4 mol dmm3 in PADAP. In the

absence of acetonitrile in the run buffer PADAP


precipitated. Chelation of metals at various concentrations was performed using a 1 X 10m4 mol dm-
solution of PADAP in acetonitrile-water
(l:l, v/v).
The chelate was readily formed at room temperature
before being injected into the capillary. The pH of
the run buffer was adjusted by the addition of 0.1
mol dm- 3 NaOH. A separation voltage of 25 kV
was applied to the capillary at an oven temperature
of 25C. All samples were introduced onto the capillary using a 30 s hydrodynamic
injection. Reproducibilities
were calculated from the results of 5
consecutive injections. Limits of detection (LODs)
for CZE studies were taken at the concentration

when the signal of the sample was three times the


peak to trough noise.
2.6, UV digestion
Stock solutions of 1 X lo- 2 mol dm- 3 nicotinamide and pyridoxine, and 1 X low3 mol dm- 3
riboflavin,
ascorbic acid, cyanocobalamin
and thiamine hydrochloride were prepared in Milli-Q water,
while retinol was made up in methanol at a 1 X lo- 3

loo

DA.

Oxspring et al./Analytica

tl.0

Chimica Acta 323 (1996) 97-105

PADAP before being injected into the capillary. The


same procedure was carried out for the detection of
cobalt in cyanocobalamin in multivitamin mixtures
formulated using vitamin stock solutions as prepared
above and containing varying concentrations of vitamin B,,.

3. Results and Discussion


3.1. UV-visible spectrophotometrk study of the
acid-base behaviour of PAN and PALMP

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.

mol dm- 3 before being combined to form a multivitamin mixture. W


digestion of 10 cm3 of
cyanocobalamin (10T4 mol drne3) solution was carried out by addition of 100 ~1 of 30% H,O, before
being W digested for 90 min at a temperature of
90C. A total volume of 6 cm3 remained after digestion of which 1.0 cm3 of W digested cyanocobalamin was added to 0.5 cm3 of 1 X 10e4 mol drnw3

The W-visible spectrophotometric behaviour of


0.2 X 10e4 mol dm- 3 (PAN)(I) was studied over the
pH range O-13. Two particular changes in the spectral behaviour were shown in the pH regions O-5
and lo- 13 as shown in Fig. 1. Plots of absorbance
vs pH at the three wavelengths 438,466 and 470 nm
yielded ply, values of 2.5 and 11.2. These values
correspond to the pyridinium ion and the phenolic
group, respectively. The predicted p K, value for the
pyridinium ion [12] is given by the equation
pK, = 5.25 - 5.90Ca

1.4.

1.2 -

1.0 -

0.6 -

0.6 -

Fig. 2. UV-visible

spectrophotometric

behaviour

of the four different absorbing

forms of PADAP, i.e., H,A* +, H2A+,

HA and A-,

DA. Oxspring et al./Analytica

where Cg is the sum of the Hammett subs&tent


constants. In the absence of a value in tables [12] for
the appropriate substituent as in I, a value of 0.82 for
the o-substitution of N=N-(C6H,-2-OH)
in a phenol was used 1121 and this yielded a predicted pK,
value of 0.41 not too far removed from the actual
pK, value of 2.5. The pK, value corresponding to
ionisation of the phenolic group is higher than that of
phenol itself (pK, = 9.9) [12]. This is not surprising
since such o-hydroxyazo compounds are capable of
intramolecular hydrogen bonding to form 6-membered rings as shown in II and hence are more
difficult to ionise than phenol itself.

101

Chimica Acta 323 (1996) 97-105

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.

Furthermore, such o-hydroxyazo compounds in these


6-membered ring structures (II) give rise to tautomers with imine and quinone groups (i.e., hydrazones), the overall molecule absorbing at longer
wavelengths than I with azo and phenolic functional
groups (i.e., the azo tautomer). It is therefore not
unreasonable to assign the major absorption bands at
ca. 430 and 475 nm in Fig. 1 to azo and hydrazone
tautomers, respectively.
Having established pK, values and charge states
of the structurally similar model ligand I at particular
pH values, PADAP was then subjected to a similar
investigation. The W-visible
spectrophotometric
behaviour of 0.2 X 10e4 mol dme3 PADAP was
studied over the pH range 0- 14 using Britton-Robinson buffers. Fig. 2 shows the spectral behaviour of
the four PADAP species H, A*, H, A+, HA and
A-. pK, values corresponding to the various equilibria are estimated
by application
of the
Henderson-Hasselbach equation to the plot of absorbance vs. pH at wavelengths 448, 510 and 534
nm (Fig. 3). A pK, of 1.0 corresponds to the
3-bromopyridinium ion which is to be expected when
compared to related ligand (I) since a 3-Br substituent on pyridine will lower the pK, value by
several units f12]. The pK, value of 3.0 corresponds
to the iV,N-diethylanilinium ion. This is in good

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

DA. Oxspring et al./Analytica

Chimica Acta 323 (1996) 97-105

The spectra at pH 2 and 5 all show an absorption


at ca. 560 nm which could be due to such an imine.
The three pK, values at 1.0, 3.0 and 11.2 show
agreement with those established for the not dissimilar ligand 5-Br-DMPAP by Shibata et al. [14].
3.2. W-visible

spectrophotometric study of the


chelation of PADAP with Co(U), Ni(II), Cu(II) and
PbfII)

Substituted azobenzenes can chelate metal ions


using o-hydroxy or o-amino azo groups to give
square planar, tetrahedral or octahedral complexes.
PADAP reacts with a range of metal ions (e.g., Co,
Ni, Zn, Cd, Mn, Cu, Pd, V, U) to give chelates with
high molar absorptivities and large bathochromic
shifts [I]. In the case of PADAP, the pyridyl nitrogen
atom can also be used in chelation processes to give
complexes which can have a 1:l or 1:2 metal-toligand stoichiometry (Fig. 4). A 1:1 stoichiometry is
indicative of square planar and tetrahedral complexes
with three donor atoms from PADAP being involved
(the pyridine nitrogen, the @azo nitrogen and the
o-hydroxy oxygen atom) and a monodentate ligand
such as H,O from the solvent/buffer. A 1:2 stoichiometry is indicative of an octahedral complex as
is observed with Co(III)-PADAP (Fig. 4).
The UV-visible spectrophotometric behaviour of
Co(B), Ni(II), Cu(I1) and Pb(II), chelated to uncharged PADAP in pH 9 buffer, is illustrated in Fig.
5. Co(B), Ni(II), Cu(II) and Pb(I1) all shift the main
PADAP peak due to a r + m * transition at ca. 450
nm some 100 nm further into the visible to give
purple (CdII))
and red (for Ni(II), C&I) and Pb(I1))
complexes. The CdIB) complex with its double
peak at ca. 550 and 580 nm remains essentially
unchanged in the pH region O-14. This would suggest a relatively high stability constant with protonation processes discussed for the free PADAP ligand
spectrally unobserved for the CdBI)-PADAP complex in the pH range O-l 4. There is evidence of a
small absorbance change for the 580 nm peak at pH
2-3, presumably correlating with the N,N-diethylanilinium ion, which is not involved in the chelation
process. The other PADAP complexes are more
labile than that of Cd10 when studied over the pH
range O-14. For example the Cu(I1) and Pb(II) complexes break up when the pH exceeds the pK, value

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.

of the phenol group and only the spectrum of the


free ligand is observed.
3.3. Adsorptive stri+ping voltammetry of Co(H),
Cd(H), Ni(II), Cu(II), Zn(II) and Pb(IIJ chelates

Table 2 shows the reduction potentials of the


metal chelates. Only four out of the six chelates
could be determined by AdSV with the Ni(I1) chelate
giving a it-reproducible signal and the Cu(I1) chelate
giving no signal at all. The CdII), Cd(B) and Pb(I1)
chelates are reduced at similar potentials ( - 670 mV,
- 620 mV and -580 mV, respectively) and the
Zn(II) chelate has a reduction potential of - 1.071
mV, well separated from the previous three. Table 2
also shows that AdSV using the HMDE is a very
sensitive technique with the LOD for the 4 metal
chelates being in the nanomolar range. When 1 X
1O-7 mol dm- 3 Cc(B) was added to the supporting
electrolyte containing 10m6 mol dmm3 PADAP the
ligand response at -620 mV was shifted to -600
mV and a new signal appears at -670 mV which
increases with increasing Cd10 concentration. The
magnitude of the Co(III) chelate voltammetric response is not affected by the presence of equimolar
concentrations (1 X 10s7 mol drnm3) of Zn*+, Cd*+
and Pb+. A LOD of 5.0 X 10e9 mol dme3 was

DA. Oxspring et al./Adytica

Chimica Acta 323 (1996) 97-10s

103

Fig. 5. IJV-visible spectrophotometric


behaviour of PADAP (2 X 10m5 mol dmm3, using right hand ordinate) and its Co(U), Ni(II), Cu(I1)
and Pb(I1) chelates (1 X lo- mol dm- 3 using left hand ordinate) at pH 9. (-) Ni; (- - -) Co; (- - -1Pb; (- - -) PADAP; (- - - -) Cu.

tion of the chelates during CZE separations. The


Co(II), Cd(U), Ni(II), Cu(I1) and Zn(I1) chelates
could be detected in the pH range 7.7-5.0.
The
detectable chelates migrated in the elution order
Cd(II), Co(II), Ct.@), Z&I) and Ni(I1) at pH 6.0
(Fig. 6). The b e h aviour of the six metal chelates was
studied over the pH range 7.7-5.0, and showed that
the behaviour of the metal chelates varied with PH.
For example the Cd10 chelate is stable throughout
the pH range, while the Cd(I1) and Z&I) chelates
give broad peaks at the higher pH values than 6.0
and sharp peaks at pH 6.0. The Ni(I1) chelate gives a
broad peak throughout the pH range and the peak

achieved and the calibration


plot of ln(peak ht.)
versus ln(conc.) was linear and yielded a correlation
coefficient of 0.9908 (Table 2). The relative standard
deviation was calculated using the peak height of
five separate vohammograms
of a solution containing 1 X lo- mol dm- 3 Co(U) and was found to be
3.5%.
3.4. Capillary electrophoresis of Co(ZZ), Cd(ZZ),
Ni(ZZ), Cu(ZZ), Zn(ZZ)and Pb(ZZ) chelates
PADAP was added to the run buffer at a concentration of 1 X low4 mol dm-3 to prevent dissocia0.01159

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

DA. Oxspring et al./Analytica

Chimica Acta 323 (1996) 97-IO5

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)

Calibration range, 1 X lo-


mol dm- 3 down to

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

the opposite and give broad signals at high pH and


an intense signal at the lower pH (pH 6.0). The
LODs for Cu(II> and Co(E) were estimated at pH
7.0. These results are presented in Table 1 where it
can be seen that Co(B) has the lowest LOD of
5 X lo- mol dmb3. Calibration plots of peak area
versus concentration show a good adherence to linearity with correlation coefficients of 0.9905 or better (Table I>.
3.5. Determination
multivitamin mix

of cobalt in vitamin B,, and in a

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

DA. Oxspring et al./Analytica

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

Chimica Acta 323 (1996) 97-105

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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