B12 Spectrophotometry.pdf

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UV-visible spectrophotometric, adsorptive stripping

voltammetric and capillary electrophoretic study of

2-( S-bromo-2’-pyridylazo) -5diethylaminophenol

and its chelates

ANALYTICA

CHIMICA

ACTA

Analytica Chimica

Acta 323 (1996) 97-105

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-diethyl-

aminophenol (PADAP), has pK, values of 1 O, 3.0 and 11.2 corresponding to the 3-bromopyridinium ion, the NJ-diethyl-

anilinium 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; Stripping voltammetry; Electrophoresis; 2-(5’-Bromo-2’-pyridyiazo)-5-diethykrninopheno~

(PADAP); Cobalt; Vitamin B,,

1. Introduction

* Corresponding author.

The azo dyes comprise the largest group of or-

ganic reagents

used

in spectrophotometric analysis

0003-2670/ / 15:00 0 1996 Elsevier Science B.V. All rights reserved

SSDI

0003-2670(95)00624-9


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98

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

and include such reagents as PAN, PAR and Arse-

nazo III. 2-(S-Bromo-2’-pyridylazo)5-diethyl-

aminophenol (PADAP) is a derivative of PAN and

PAR and forms the basis of highly sensitive methods

for trace metal determination, with a molar absorp-

tivity (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 voltam-

metry (AdSV), e.g., bismuth 121, chromium(II1) 131,

titanium(IV) [4] copper(B) [5], iron(II1) [6] and vana-

dium(V) [7] have been determined this way. Individ-

ual metal determinations were reported down to lim-

its 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 fluo-

resence 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 pre-

sent paper is concerned with (a) a W-visible spec-

trophotometric study of the acid-base behaviour of

PADAP and the related model ligand I-(2-pyri-

dylazo)-2-naphthol (PAN) in order to establish the

charge states of PADAP at particular pH values; (b)

a W-visible spectrophotometric study of the chela-

tion 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 Cd110n vitamin B ,2

(cyanocobalamin) following destruction of the corrin

ring system by a

W

digestion procedure and chela-

tion 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 evalu-

ated.

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 auto-

matically and sequentially process up to 10 samples.

A three-electrode system was used throughout con-

sisting 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 instru-

ment (Thermo Separation Products, Stone, UK) fit-

ted 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 intro-

duced into the capillary by hydrodynamic injection

and monitored using a W-visible diode array detec-

tor (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 Hewlett-

Packard 8451 diode array spectrophotometer (Hewlett

Packard, Palo Alto, CA).

2.2. Reagents and analytes

AnalaR grade nitrate, chloride, and sulfate salts

were used for preparing standard solutions of the

metal ions (BDH, Poole, UK). The buffers and elec-

trolytes used were all of AnalaR grade and pur-


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DA. Oxspri ng et al./Analytica Chimica Acta 323 (1996) 97-105

99

chased from BDH. The ligands 2-(S-bromo-2’-pyri-

dylazo)J-diethylaminophenol (PADAP) and l-(2-

pyridylazo)-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 acetonitrile-

water (1: 1, v/v). The vitamins cyanocobalamin

(B ,* 1, riboflavin (B,), pyridoxine (B6), nicotinamide

(B,), thiamine hydrochloride (B r ), ascorbic acid (0

and retinol (A) were also purchased from Aldrich.

All solutions were made up in Mini-Q 18 Q cm

water (Millipore).

2.3. Procedures

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

pH range 2- 12 was covered by stock Britton-Robin-

son buffer (0.02 mol dme3) with the pH adjusted by

the 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 con-

stant 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 car-

ried 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

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 in-

creased 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.

2.5. CZE procedure

Prior to use the fused silica capillary was washed

through with methanol for 5 min at 40°C followed

by 0.1 mol dmw3 sodium hydroxide and Mini-Q

water for 5 min at 60°C. The capillary was finally

washed through with run buffer for 5 min at 25°C.

Separation was performed with a run electrolyte of

0.05 mol dmm3 sodium acetate, 20 (v/v) in ace-

tonitrile and 1 X 10e4 mol dmm3 in PADAP. In the

absence of acetonitrile in the run buffer PADAP

precipitated. Chelation of metals at various concen-

trations 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 25°C. All samples were introduced onto the capil-

lary using a 30 s hydrodynamic injection. Repro-

ducibilities 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 nicoti-

namide and pyridoxine, and 1 X low3 mol dm- 3

riboflavin, ascorbic acid, cyanocobalamin and thi-

amine hydrochloride were prepared in Milli-Q water,

while retinol was made up in methanol at a 1

X lo- 3


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loo

DA. Oxsprin g et al./Analytic a Chimica Acta 323 1996) 97-105

tl.0

A

2

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 multivi-

tamin mixture. W digestion of 10 cm3 of

cyanocobalamin (10T4 mol drne3) solution was car-

ried out by addition of 100 ~1 of 30 H,O, before

being W digested for 90 min at a temperature of

90°C. A total volume of 6 cm3 remained after diges-

tion of which 1.0 cm3 of

W

digested cyanocobal-

amin was added to 0.5 cm3 of 1

X

10e4 mol drnw3

Fig. 2.

UV-visible spectrophotometric behaviour of the four different absorbing forms of PADAP, i.e., H,A*

+, H2A+, HA and A-,

A 1.4.

1.2

1.0

0.6

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 vita-

min B,,.

3.

Results and Discussion

3.1.

UV-v isibl e spectrophotometr k study of the

acid-base behavi our of PAN and PALM P

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 spec-

tral 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

0.6 -


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DA. Oxspri ng et al./Analytica Chimica Acta 323 (1996) 97-105 101

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 phe-

nol 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-mem-

bered rings as shown in II and hence are more

difficult to ionise than phenol itself.

Furthermore, such o-hydroxyazo compounds in these

6-membered ring structures

II)

give rise to tau-

tomers with imine and quinone groups (i.e., hydra-

zones), 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-Robin-

son 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 equi-

libria are estimated by application of the

Henderson-Hasselbach equation to the plot of ab-

sorbance 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 sub-

stituent 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

0.8

1 0.6

s

s

0.4

~0 ‘1-1-1’1.1-1’1.1.1.1’1.1-1’(

0

1

2 3 4 5 6 7 8

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 calcu-

lated 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 corre-

sponds to the phenolic group.

The W-visible spectra again illustrate the exis-

tence of tautomeric equilibria with the two major

absorptions at ca 450 nm and ca. 520 nm presumably

representing azo and hydrazone tautomers, respec-

tively. 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-mem-

bered ring structure is broken down, the 520 nm

absorption band representing the hydrazone is pre-

dominant. The W-visible spectra for PADAP (III)

are further complicated by tautomeric equilibria in-

volving the NJ-diethylamino group where imine

tautomers such as (IV) have been proposed [ 131.


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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 dissimi-

lar ligand 5-Br-DMPAP by Shibata et al. [14].

3.2.

W-vi sible spectrophotometr ic study of the

chelat ion of PAD AP w it h Co U), N i 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-to-

ligand 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 stoi-

chiometry is indicative of an octahedral complex as

is observed with Co(III)-PADAP (Fig. 4).

OP .

I

0

1

2

3

4

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; ( copper.

of the phenol group and only the spectrum of the

free ligand is observed.

3.3.

Adsorpti ve stri +ping vol tammetr y of Co H),

Cd H), N i II ), Cu II ), Z n II ) and Pb II J chelat es

The UV-visible spectrophotometric behaviour of Table 2 shows the reduction potentials of the

Co(B), Ni(II), Cu(I1) and Pb(II), chelated to un-

metal chelates. Only four out of the six chelates

charged PADAP in pH 9 buffer, is illustrated in Fig. could be determined by AdSV with the Ni(I1) chelate

5. Co(B), Ni(II), Cu(II) and Pb(I1) all shift the main giving a it-reproducible signal and the Cu(I1) chelate

PADAP peak due to a r + m * transition at ca. 450

giving no signal at all. The CdII , Cd(B) and Pb(I1)

nm some 100 nm further into the visible to give

chelates are reduced at similar potentials ( - 670 mV,

purple CdII))nd red (for Ni(II), C&I) and Pb(I1)) - 620 mV and -580 mV, respectively) and the

complexes. The CdIB) complex with its double

Zn(II) chelate has a reduction potential of - 1.071

peak at ca. 550 and 580 nm remains essentially

mV, well separated from the previous three. Table 2

unchanged in the pH region O-14. This would sug-

also shows that AdSV using the HMDE is a very

gest a relatively high stability constant with protona-

sensitive technique with the LOD for the 4 metal

tion processes discussed for the free PADAP ligand chelates being in the nanomolar range. When 1 X

spectrally unobserved for the CdBI)-PADAP com- 1O-7 mol dm- 3 Cc(B) was added to the supporting

plex in the pH range O-l 4. There is evidence of a electrolyte containing 10m6 mol dmm3 PADAP the

small absorbance change for the 580 nm peak at pH ligand response at -620 mV was shifted to -600

2-3, presumably correlating with the N,N-diethyl- mV and a new signal appears at -670 mV which

anilinium ion, which is not involved in the chelation increases with increasing Cd10 concentration. The

process. The other PADAP complexes are more magnitude of the Co(III) chelate voltammetric re-

labile than that of Cd10 when studied over the pH sponse is not affected by the presence of equimolar

range O-14. For example the Cu(I1) and Pb(II) com- concentrations (1

X

10s7 mol drnm3) of Zn*+, Cd*+

plexes break up when the pH exceeds the pK, value and Pb’+. A LOD of 5.0 X 10e9 mol dme3 was


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DA. Oxspri ng et al./Adytica Chimi ca 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.

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 contain-

ing 1 X lo-’ mol dm- 3 Co(U) and was found to be

3.5 .

3.4. Capil l ary el ectrophor esis of Co ZZ ), Cd ZZ),

Ni ZZ ), Cu ZZ), Zn Z Z) nd Pb ZZ) helat es

PADAP was added to the run buffer at a concen-

tration of 1 X low4 mol dm-3 to prevent dissocia-

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 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 Cd10chelate 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

0.01159

58.20

$

B

::

6.39

9

4.96

I

-0.oco61

I

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.


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DA. Oxspri ng 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

Calibration range, 1 X lo-“ Correlation

(mol dmm3) mol dm- 3 down to

coefficient (n = 4)

cd10

587 7.0 1.6 X 10’ 5 x 10-7 5 x 10-7 0.9982

Cd(R) 548 6.0 6.6 x lo4 5 x 10-6 5 x 10-6 0.9905

C&II) 554 7.0 1 x106 5 x 10-e 5 x 10-6 0.9910

Ni(I1) 556 7.7 2.8 X lo* 1 x 10-6 1 x 10-6 0.9988

Pb(II) 576 1.7 8.6 x 103 1 x 10-6 1 x 10-6 0.9958

Z&I) 548 6.0 2.5 X 10’ 1 x 10-s 1 x 10-6 0.9991

area of the Pb(I1) chelate decreases with pH de- the opposite and give broad signals at high pH and

crease. The efficiency in terms of number of plates an intense signal at the lower pH (pH 6.0). The

N where N = 16 (t,/W>* was calculated for the

LODs for Cu(II> and Co(E) were estimated at pH

individual metal chelates (Table 11, which showed

7.0. These results are presented in Table 1 where it

that the Co, Cd, Cu and Zn chelates have high

can be seen that Co(B) has the lowest LOD of

efficiencies in the 6.6

x

IO4 to 1 X lo6 range. The

5

X

lo-’ mol dmb3. Calibration plots of peak area

Pb and Ni chelates have relatively poor efficiency

versus concentration show a good adherence to lin-

values of 8.6

X

lo3 and 2.8

X

lo* respectively due earity with correlation coefficients of 0.9905 or bet-

to their poor peak shape.

ter (Table I>.

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 stoi-

chiometry). 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).

3.5. Determi nation of cobalt in vitamin B,, and in a

multivitamin mix

Co(I1) will complex with PADAP to form a par-

ticularly stable chelate which can be detected and

determined selectively by AdSV and CE. Both tech-

niques were compared for their ability to determine

Co(II1) in vitamin B,, (cyanocobalamin) following

destruction of the corrin ring system by UV diges-

tion using H,O,.

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

Table 2

AdSV conditions for detection and determination of selected

metal ions using PADAP, LOD values and correlation coefficients

Metal Reduction peak

LOD (X lo-* Correlation

ion potential of PADAP mol dm-‘) coefficient

chelate (mVI from logtpeak

ht.) - logkonc.)

plots (n = 6)

cd10 -670

0.5

0.9908

Cu(II) -

Cd(R) - 620

8.3 0.9944

Zn01) - 1.071

4.1

0.9999

Ni(II)

-

PdII) -580

3.0 0.9954


9/9

DA. Oxspri ng et al./Analytica Chimica Acta 323 (1996) 97-105

105

Using the methodology outlined in the Experi-

mental 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 cali-

bration 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 diges-

tion procedure and had been subsequently chelated

with PADAP. Linear regression analysis on the cali-

bration plot over the concentration range 1

X

10e4-

5

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 concentra-

tions 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 recov-

ery. 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

charge states of PADAP at various pH values. Deter-

mination 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 equimo-

lar and higher concentrations of other vitamins found

in multivitamin mixtures following UV digestion.

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