J. Braz. Chem. Soc., Vol. 16, No. 6A, 1154-1159, 2005.

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Article

Study on the Determination of Lead, Cadmium, Mercury, Nickel and Zinc by a Rapid
Column High-Performance Liquid Chromatography
,a a,b a,b a,b
Guangyu Yang* , Qiufen Hu , Zhangjie Huang and Jiayuan Yin
a
Department of Chemistry, Yunnan University, Kunming, 650091, P.R. China
b
Department of Chemistry, Yuxi Teacher’s College, Yuxi, 653100, P.R. China

Um novo método para determinação simultânea de cinco íons de metais pesados em água, alimentos
e cabelo, usando-se cromatografia líquida de alta eficiência foi desenvolvido. Os íons de chumbo, cádmio,
mercúrio, níquel e zinco foram derivatizados (pré-coluna) com 2-(2-quinolinil-azo)-4-metil-1,3-
dihidroxidobenzeno (QAMDHB) para formar quelatos coloridos. A seguir, os quelatos Pb-QAMDHB,
Cd-QAMDHB, Hg-QAMDHB, Ni-QAMDHB e Zn-QAMDHB foram enriquecidos pela extração
em fase sólida, com cartucho de C18. O fator de enriquecimento de 100 foi obtido ao eluir os quelatos
retidos no cartucho, com tetrahidrofurano (THF). Esses quelatos foram separados em uma coluna de
análise rápida ZORBAX Stable Bound (4,6×50 mm, 1,8 μm) com 68% de metanol (contendo 0,05 mol
L-1 da solução tampão de ácido acético-pirrolidina, pH 8,5, e 0,01 mol L-1 de CTMAB) como fase móvel,
fluxo de 2,0 mL min-1, em um detector por um detector de arranjo de fotodiodos a 450 ~ 600 nm. Os
quelatos Pb-QAMDHB, Cd-QAMDHB, Hg-QAMDHB, Ni-QAMDHB e Zn-QAMDHB foram
separados completamente em 2,0 min. Os limites de detecção do chumbo, cádmio, mercúrio, níquel e
zinco são 1,2 ng L-1, 1,0 ng L-1, 0,8 ng L-1, 1,6 ng L-1 e 1,9 ng L-1, respectivamente, nas amostras originais.
Este método foi aplicado na determinação dos cinco íons desses metais pesados com bons resultados.

A new method for the simultaneous determination of five heavy metal ions in water, food and
human hair by a rapid column high-performance liquid chromatography was developed. The lead,
cadmium, mercury, nickel and zinc ions were pre-column derivatized with 2-(2-quinolinylazo)-4-
methyl-1,3-dihydroxidebenzene (QAMDHB) to form colored chelates. Then the Pb-QAMDHB, Cd-
QAMDHB, Hg-QAMDHB, Ni-QAMDHB and Zn-QAMDHB chelates were enriched by solid
phase extraction with C18 cartridge. The enrichment factor of 100 was achieved by eluting the retained
chelates from the cartridge with tetrahydrofuran (THF). These chelates were separated on a ZORBAX
Stable Bound rapid analysis column (4.6×50 mm, 1.8 μm) with 68% methanol (containing 0.05 mol
L-1 of pH 8.5 pyrrolidine-acetic acid buffer solution and 0.01 mol L-1 of CTMAB) as mobile phase at
a flow rate of 2.0 mL min-1 using a photodiode array detector from 450-600 nm. The Pb-QAMDHB,
Cd-QAMDHB, Hg-QAMDHB, Ni-QAMDHB and Zn-QAMDHB chelates were separated
completely within 2.0 min. The detection limits of lead, cadmium, mercury, nickel and zinc are 1.2 ng
L-1, 1.0 ng L-1, 0.8 ng L-1, 1.6 ng L-1 and 1.9 ng L-1, respectively in the original samples. This method
was applied to the determination of these five heavy metal ions with good results.

Keywords: rapid column high-performance liquid chromatography, 2-(2-quinolinylazo)-4-

methyl-1,3- dihydroxidebenzene, heavy metal ions

Introduction
The RP-HPLC technique with pre-column In previous work, Many azo dyes, such as pyridylazo
derivatization has been proved to be a favorable and reagents, thiazolylazo reagents, benzothiazolylazo
reliable technique for the separation and determination of reagents and the like have been used as chelating reagent
trace amount of metal ions. Many kinds of reagents have for the determination of metal ions by RP-HPLC. 9-16
been examined as pre-column derivatization reagents, and However, a long separation time (more then 10 min) is
several review articles have appeared on this approach.1-8 needed for the routine chromatographic methods. To
shorten the separation time, in this paper, 2-(2-
* e-mail: huqiufena@163.com quinolinylazo)-4-methyl-1,3-dihydroxidebenzene
Vol. 16, No. 6A, 2005 Study on the Determination of Lead, Cadmium, Mercury, Nickel and Zinc 1155

(QAMDHB) was used as pre-column derivatization

reagents for lead, cadmium, mercury, nickel and zinc, and
a ZORBAX Stable Bound rapid analysis column (4.6×50
mm, 1.8 μm) was used for the separation of metal-QAMDHB
chelates. The five chelates were separated completely
within 2.0 min in this method. The separation time was
greatly shortened compared to the routine chromatographic
methods. Based on this, a rapid, sensitive and selective
method for the simultaneous determination of the five
heavy metal ions in water, food and human hair was
developed.

Experimental Figure 1. The infrared spectrum of QAMDHB.

Synthesis of QAMDHB

2-Aminoquinoline (7.2 g, 0.052 mol) was dissolved in

a 500 mL anhydrous ethanol. To which, sodamide (2.0 g,
0.051 mol) was added and the mixture was refluxed in
boiling water bath for 5 h, followed by the addition of
isoamyl nitrite (7.4 mL, 0.052 mol). The solution was
refluxed for 30 min with a boiling water bath, and then the
solution was cooled and placed over night under 0 oC. The Figure 2. The 1H nuclear magnetic resonance spectrum of QAMDHB.
diazo solution was obtained by filtering this solution with
an isolation yield of 95%. Thereafter, the diazo solution detector (Waters Corporation, USA). The pH values were
was dissolved in 200 mL anhydrous ethanol, followed by determined with a Beckman Φ-200 pH meter. The
the addition of 4-methyl-1,3-dihydroxidebenzene (5.8 g, separation column used was a ZORBAX Stable Bound
0.047 mol). The carbon dioxide was ventilated into the rapid column (4.6×50 mm, 1.8 μm) (Agilent Corporation,
solution with stirring until the pH reaches about 8.0. The USA). The cartridge used is Zorbax C 18 solid phase
solution was stood for two days, and then the solution was extraction cartridge (1 cm3 per 50 mg, 30 μm) (Agilent
diluted with 400 mL water. The QAMDHB in diluted Corporation, USA). The extraction was performed on Waters
solution was extracted with chloroform. The chloroform Solid Phase Extraction (SPE) Device (The device can
was evaporated and the residue was re-crystallized with prepare twenty samples simultaneously).
30% ethanol. QAMDHB was obtained with 35% yield. All of the solutions were prepared with ultra-pure water
The structure of QAMDHB was verified by elemental obtained from a Milli-Q50 SP Reagent Water System
analysis [C15H11N3O2, found (calculated): C 68.8 (68.1), N (Millipore Corporation, USA). QAMDHB was dissolved
15.1 (14.8), H 5.06 (4.98)]. IR (Figure 1) (KBr) νmax(cm-1): with THF to make a 1.0×10-4 mol L-1 of solution. Pb(II),
3580 (νO-H); 1615, 1565, 1530, 1429 (νC=C, N=N); 2925, 2873 Cd(II), Hg(II), Ni(II) and Zn(II) standard solution (1.0 mg
(νC-H); 1460, 1425, 1380 (δC-H); 3050, 3025 (σAr-H); 1050 mL-1) was obtained from the Chinese Standards Center,
(νC-O); 1175, 1120, 865, 785, 750 (δAr-H). 1H NMR (Figure and a working solution of 0.5 μg mL-1 was prepared by
2) (solvent: d6-acetone) (δ ppm): 6.88-7.65 (m 8 H, Ar-H); diluting this standard solution. The methanol and
5.34, 5.46 (σ 1H, Ar-OH); 2.22 (σ3H, Ar-CH3); MS (Figure tetrahydrofuran (THF) used were HPLC grade (Fisher
3): 279 (M+). Corporation, USA). Pyrrolidine-acetic acid buffer solution
(0.5 mol L-1, pH 8.5) and CTMAB solution (1%, dissolving
Chemicals and apparatus with 10% ethanol) was used. The mobile phase used was
68% methanol (containing 0.05 mol L -1 of pH 8.5
The HPLC system was consisted of a Waters 2690 pyrrolidine-acetic acid buffer solution and 0.01 mol L-1 of
Alliance separation model and a 996 photodiode array CTMAB). All other reagents used were of analytical
1156 Yang et al. J. Braz. Chem. Soc.

Figure 3. The mass spectrum of QAMDHB.

reagent-grade. The glass and Teflon wares used were soaked Figure 4. Chromatogram of standard sample and real sample: (1)
Rice sample, (2) Standard sample. Injection volume 10 mL. The
in 5% of nitric acid for a long time, and then thoroughly
concentration of Pb, Cd, Hg, Ni, Zn is 20 mg L-1 in standard sample.
wash with pure water. Detection wavelength is 555 nm. Other conditions as in standard
procedure.
Standard procedure
reagents. So the amount of QAMDHB must in excess. In
An appropriate volume of sample solution or standard this experiment, 8.0 mL of 1.0×10-4 mol L-1 QAMDHB
solution was transferred into a 100 mL of volumetric flask. was sufficient to chelate the ions in real samples, and 8.0
To which, 8.0 mL 1.0×10-4 mol L-1 of QAMDHB THF mL QAMDHB solution was recommended.
solution, 10 mL pH 8.5 pyrrolidine-acetic acid buffer The experiments show that in the nonionic surfactants
solution and 2.0 mL of 1% of CTMAB solution were and cationic surfactants medium, the sensitivity of the
added. The solution was diluted to the volume with water metal-QAMDHB chelates was increased markedly. Various
and mixing well. After 10 min, the solution was passed nonionic surfactants and cationic surfactants enhance the
through the C18 cartridge at a flow rate of 10 mL min-1. absorbance in the following sequence: CTMAB > Tween-
When the enrichment had finished, the retained chelates 80 > CPB > Tween-20 > TritonX-100. Therefore, CTMAB
were eluted from the cartridge with 1.0 mL of THF at a was selected as additive in this experiment. The use of 1 ~
flow rate of 5 mL min-1 in an opposite direction. The 4 mL of CTMAB solution give a constant and maximum
solution was filtered with 0.45 mm of filters and adjusted absorbance in this experiment. Accordingly, 2.0 mL
to the volume of 1.0 mL. 10 μL of sample was injected for CTMAB solution was recommended.
HPLC analysis. A tridimensional (X axis: retention time, Y
axis: wavelength, Z axis: absorbance) chromatogram was Solid phase extraction
recorded from 450~650 nm with photodiode array detector
and the chromatogram of 555 nm was shown in Figure 4. Both the enrichment and the elution were carried out
. on a Waters SPE device, which can prepare twenty samples
Results and Discussion simultaneously, the flow rate was set to 10 mL min-1 when
enrichment and 5 mL min-1 when elution.
Precolumn derivation Some experiments were carried out in order to
investigate the retention of metal-QAMDHB chelate on
The optimal pH for the QAMDHB reacts with metal the cartridge. It was found that the Pb-QAMDHB, Cd-
ions is 7.5 ~ 11.2 for lead, 7.8 ~ 12.5 for cadmium, 8.0 ~ QAMDHB, Hg-QAMDHB, Ni-QAMDHB and Zn-
11.8 for mercury, 4.8 ~ 9.2 for nickel and 7.2 ~ 9.4 for zinc. QAMDHB chelates could be retained on cartridge
Therefore, a 0.5 mol L-1 of pyrrolidine-acetic acid buffer quantitatively when they pass the cartridge as aqueous
solution (pH 8.5) was recommended to control pH. solution. The capacity of the cartridge for QAMDHB was
It was found that 1.0 mL of 1.0×10-4 mol L-1 QAMDHB 30 mg and for its metal-QAMDHB chelate was 25 mg in a
THF solution was sufficient to chelate 10.0 μg of Pb(II), 100 mL of solution. In this experiment, the cartridge has
Cd(II), Hg(II), Ni(II) and Zn(II) ions. But in the real samples, adequate capacity to enrichment the metal-QAMDHB
the foreign ions can react with QAMDHB to consume chelate and the excessive QAMDHB.
Vol. 16, No. 6A, 2005 Study on the Determination of Lead, Cadmium, Mercury, Nickel and Zinc 1157

Various organic solvents were studied as eluant for the chromatographic separation time, A ZORBAX Stable
eluting the retained QAMDHB and its metal-chelate from Bound rapid analysis column (4.6×50 mm, 1.8 μm) was
the cartridge. It was found that the THF, acetone, selected in this experiment. With rapid analysis column,
acetonitrile, ethanol and methanol could elute the the five chelates were separated completely within 2
QAMDHB and its metal-chelate from cartridge min. Compared to the routine chromatographic method,
quantitatively. For completely eluting the metal- more then 80% of separation time was shortened.
QAMDHB chelates from cartridge, the volume of the
solvent needed is THF 0.9 mL, isopentyl alcohol 1.4 mL, Spectrophotometric properties
acetone 1.7 mL, acetonitrile 1.9 mL, ethanol 2.2 mL,
methanol 2.4 mL. The maximal enrichment factor was The absorption spectrum of metal-QAMDHB
achieved when THF was selected as eluant. Therefore, chelates was obtained by measured with a Shimidzu UV-
THF was selected as eluant in this experience. The metal- 2401 spectrophotometer. The absorption data were
QAMDHB chelate has a good stability in weak alkaline given in Table 1. In this experiment, to get maximum
medium. The eluant containing a 0.05 mol L -1 sensitivity, each metal-QAMDHB chelates was
pyrrolidine-acetic acid buffer (pH 8.5) can increase the monitored at its maximum absorption wavelength (Pb-
stability of the metal-QAMDHB chelate in the course QAMDHB 562 nm, Cd-QAMDHB 556 nm, Hg-
elution. Therefore, the THF (containing 0.05 mol L-1 QAMDHB 570 nm, Ni-QAMDHB 568 nm, Zn-QAMDHB
pyrrolidine-acetic acid buffer solution (pH 8.5)) was 548 nm).
selected as eluant in this experience. The experiment show
that it was easier to elute the retained QAMDHB and its Calibration graphs
metal-chelate in reverse direction than in forward
direction (only 0.9 mL of eluant is needed when eluted Under the optimum conditions, the regression
in reverse direction, however, 2.2 mL of eluant is needed equations of metal-QAMDHB chelates were established
when eluted in forward direction), so it is necessary to based on the standard sample injected and its peak area.
upturned the cartridge when elution. 1.0 mL of eluant The limits of detection are calculated by the ratio of signal
was sufficient for elute the QAMDHB and its metal- to noise (S/N=3). The results were shown in Table 2. The
chelate from cartridge quantitatively at a flow rate of 5 reproducibility of this method was also examined for 20
mL min-1. Therefore the volume of 1.0 mL eluant was mg L-1 of Pb(II), Cd(II), Hg(II), Ni(II) and Zn(II). The relative
selected in this experiment. standard deviations (n=9) were shown in Table 2 too.

Chromatographic separation Table 1. Spectrophotometric properties of metal-QAMDHB che-

lates

The experiments showed that the Pb-QAMDHB, Cd- Metal Pb- Cd- Hg- Ni- Zn-
chelates
QAMDHB, Hg-QAMDHB, Ni-QAMDHB and Zn-
λmax (nm) 562 556 570 568 548
QAMDHB chelates have a good stability in the presence
ε (×104 L mol-1 cm-1) 8.23 8.68 8.94 7.76 6.67
of alkaline acid buffer solution and CTMAB medium.
The pH of mobile phase within 7.8-9.4 and containing
a 0.005-0.3 mol L-1 of CTMAB in the mobile phase can Interference
avoid the metal-chelate decomposing in the course of
separation and get a good peak shape. So methanol/ Under the pre-column derivatization conditions, the
water (68/32, v/v) (containing 0.05 mol L-1 of pH 8.5 foreign ions of Fe(III), Co(II), Mn(II), Ag(I), Cu(II), V(V)
pyrrolidine-acetic acid buffer solution and 0.01 mol L-1 which can reacts with QAMDHB to form color chelates.
of CTMAB) was selected as mobile phase. To shorten To examine the selectivity of this method, the

Table 2. Regression equation, coefficient and detect limit

Components Regression Equation a Linearity Range (μg L-1) Coefficient Detect limits (ng L-1) b
RSD % (n=9)
4
Pb-QAMDHB A=5.48×10 C+158 0.7-800 r=0.9995 1.2 1.9
Cd-QAMDHB A=5.99×10 4 C-229 0.5-650 r=0.9992 1.0 2.1
Hg-QAMDHB A=6.68×10 4 C+246 0.6-600 r=0.9994 0.8 2.2
Ni-QAMDHB A=5.28×10 4 C+287 0.8-900 r=0.9992 1.6 2.4
Zn-QAMDHB A=5.15×10 4 C-229 0.6-950 r=0.9993 1.9 1.8
a
In the measured solution; b in the original digested sample.
1158 Yang et al. J. Braz. Chem. Soc.

interference of these foreign ions was investigated. procedure. The results (deducted the reagents blank)
When 5.0 mL of 1.0×10-4 mol L-1 QAMDHB was used, were shown in Table 3.
with 20 μg L-1 of Pb(II), Cd(II), Hg(II), Ni(II) and Zn(II)
respectively, the tolerance amount with an error of ±5% Applied to the water samples
was 3000 μg L-1 for Fe(III), Cu(II), Co(II), Mn(II) and
500 μg L-1 for Ag(I), V(V). This method is high selectivity. For the fresh water (tap water, river water and lake
water), the water sample was analyzed according to the
Applied to the rice and human hair samples general procedure. The results (deducted the reagents
blank) were shown in Table 4, together with the results
A sample of 0.2-0.50 g (rice and human hair) was of a recovery test by added 0.2 μg of Pb, Hg, Cd, Ni and
weighted accurately into the teflon high-pressure Zn in water sample and diluted to 100 mL of final
microwave acid-digestion bomb (Fei Yue Analytical solution. For planting effluents, the sample was digested
Instrument Factory, Shanghai, China). To which, 5.0 mL as literature 17 and analyzed according to the general
of concentrated nitric acid and 6.0 mL of 30% hydrogen procedure. The results (deducted the reagents blank)
peroxide were added. The bombs were sealed tightly were shown in Table 4 too, together with results of a
and then positioned in the carousel of the microwave recovery test by added 0.2 μg of Pb, Hg, Cd, Ni and Zn
oven (Model WL 5001, 1000 W, Fei Yue Analytical in water sample and diluted to 100 mL of final solution.
Instrument Factory, Shanghai, China). The system was A standard method using atomic absorption
operated at full power for 6.0 min. The digest was spectrometry reported in literature 17 had also been used
evaporated to near dryness. The residue was dissolved as a reference method. The results are shown in Table 5.
with 5 mL of 5% of nitric acid and transferred into a 100
mL of calibrated flask quantitatively, then diluted the Conclusions
solution to volume with water. The Pb(II), Cd(II), Hg(II),
Ni(II) and Zn(II) contents were analyzed by using a In this paper, 2-(2-quinolinylazo)-4-methyl-1,3-
proper volume of this solution according to general dihydroxidebenzene was used as pre-column

Table 3. Determination results of certified standard food samples

Samples Standard value (μg g-1) By this method (μg g-1) RSD % (n=5)

Rice As(0.285), Ba(21.2), Bi(0.342), Ca(2900), Cd(0.318),Ce(1.25), Cd(0.301), Hg(0.197),

(GBW08458) Co(4.71), Cr(3.76), Cu(10.2), Fe(54), Hg(0.186), Mg(360), Mn(22.5), Ni(3.64), Pb(0.884), Zn(8.49) 2.9
Mo(0. 735), Ni(3.83), Pb(0.852), V(2.86), Zn(8.76)
Human Hair As(0.121), Ba(11.7), Bi(0.825), Ce(0.643), Cd(0.528), Co(6.25), Cd(0.504), Hg(0.214),
(GBW08126) Cr(0.8), Cu(11.2), Fe(123), Hg(0.242), Mn(68.2), Ni(5.61), Pb(1.06), Ni(5.82), Pb(1.15), Zn(13.4) 2.7
Sn(1.24), Se(0.0412), Zn(12.7), V(3.86)

Table 4. Determination results (μg L-1) of the water sample with this method
Components Samples (μg L-1) RSD % (n=5) Recovery %
River water Lake water Plant effluent Tap water (n=5)
Pb 19.6 26.5 75.8 10.5 2.8 96
Cd 7.26 11.5 23.2 - 3.2 106
Hg 5.76 7.43 38.6 - 3.0 97
Ni 24.9 32.5 58.7 6.84 3.2 93
Zn 76.2 63.5 121 36.4 3.4 102

Table 5. Determination results (μg L-1) of the water sample with reference method
Components Samples (μg L-1) RSD % (n=5) Recovery %
River water Lake water Plant effluent Tap water (n=5)
Pb 20.6 28.4 79.6 11.3 3.4 105
Cd 6.94 10.2 21.8 - 3.6 97
Hg 5.18 6.89 39.5 - 3.8 108
Ni 26.1 33.4 57.1 7.16 3.8 92
Zn 77.9 64.3 114 37.1 3.5 96
Vol. 16, No. 6A, 2005 Study on the Determination of Lead, Cadmium, Mercury, Nickel and Zinc 1159

derivatization reagent for Pb, Hg, Cd, Ni and Zn ions, and 3. Wang, L.; Hu, Q. F.; Yang, G. Y.; Yuan, Z. B.; J. Anal. Chem.
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