Simultaneous determination of
nickel, lead, cadmium and mercury in water by RP-HPLC followed by on-line column
enrichment
Tai Xi 1, Li Deliang 1,
Li Haitao 1, Hu Qiufen 1,2, Yang Guangyu 2*, Yin Jiayuan 2
(1Department of Chemistry, Yuxi Teacher's College, Yuxi, 653100; 2
Department of Chemistry, Yunnan University, Kunming, 650091,China)
Received Dec. 23, 2003; Supported by Natural
Science Foundation of Yunnan Province (0111143) and Yuxi Teacher's College (02476).
Abstract A new method
for the simultaneous determination of four heavy metal ions, nickel, lead, cadmium and
mercury ions as metal-tetra-(2-aminophenyl)-porphyrin
(T2APP) chelates was developed
using reversed-phase high performance liquid chromatography (RP-HPLC) equipped with a
photodiode array detector and combined with an on-line enrichment technique. The detection
limits (S/N=3) of nickel, lead, cadmium and mercury are 3.5 ng/L, 2.5 ng/L, 3.0 ng/L and
3.0 ng/L, respectively. This method has been applied to the determination of nickel, lead,
cadmium and mercury ions in water with good results.
Keywords RP-HPLC, tetra-(2-aminophenyl)-porphyrin, on-line enrichment, heavy metal
ions
The RP-HPLC techniques with
pre-column derivatization have been proved to be a favorable and reliable technique for
the determination of trace amount of metal ions. Many kinds of reagents have been examined
as pre-column derivatization regents, and several review articles have appeared on this
approach [1-4]. Among the various kinds of reagents, porphyrin ligands are
useful because of its high molar absorptivity and high stability [5-7]. In this
paper, we report the preconcentration and separation of Ni-T2APP, Pb-T2APP,
Hg-T2APP and Cd-T2APP chelates on a Waters XterraTM RP18
column (pH 1-12) with a valve switching HPLC system equipped with a photodiode array
detector. By this system, the injection of a large volume of sample was possible and the
Ni-T2APP, Pb-T2APP, Hg-T2APP and Cd-T2APP
chelates were successfully separated. This method has been applied to the determination mg/L (ppb) level of
nickel, lead, cadmium and mercury ions in water with good results.
1. EXPERIMENTAL PROCEDURES
1.1 Apparatus
The on-line enrichment system used is shown in Fig 1, including a Waters 2690 Alliance
quadripump, Waters 515 pump, Waters 996 photodiode array detector, six-port switching
valve, large volume injector ( containing a 5.0 ml sample), enrichment column [Waters
XterraTM RP18 (5 m m, 3.9 ¡Á20 mm)] and analytical column [Waters Xterra TM RP18 (5 mm, 3.9¡Á150 mm)]. The
pH value was determined with a Beckman F-200 pH meter.
1.2 Chemicals
All of the solutions were prepared with ultra-pure water obtained from a Milli-Q50 SP
Reagent Water System (Millipore Corporation, USA). Nickel(II), lead(II), cadmium(II) and
mercury(II) standard solution of 1.0 mg/ml was obtained from the Chinese Standards Center,
and a working solution of 0.2 mg/ml was prepared by diluting this standard solution. HPLC
grade methanol and THF (Fisher Corporation, USA), pyrrolidine-acetic acid buffer solution,
0.5 mol/L, pH=10, and TritonX-100 solution, 1.0% (v/v) were used. T2APP was
synthesized in our laboratory as in the literature [8], and was dissolved with THF to make
a 1.0¡Á10-4 mol/L of solution. Mobile phase A: 0.05 mol/L pH=10
pyrrolidine-acetic acid buffer solution. Mobile phase B: methanol (containing 0.05 mol/L
pH=10 pyrrolidine-acetic acid buffer salt). Mobile phase C: acetone (containing 0.05 mol/L
pH=10 pyrrolidine-acetic acid buffer salt). All other reagents used were of analytical
reagent-grade. The glass and Teflon ware used were soaked in 5% of nitric acid overnight,
and then thoroughly washed with pure water.
Fig. 1 On-line enrichment system using the valve-switching technique
Pump A, Waters 515 Pump. Pump B, Waters 2690 Alliance quadripump. Injector can contain 5
ml of sample. Six-port switching valve (Waters Corporation). Enrichment Column, Waters
XterraTM RP18 (5mm , 3.9¡Á20). Analytical column, Waters XterraTM RP18
(5mm , 3.9¡Á150). Detector, Waters 996 photodiode array detector. MP A, 0.05
mol/L pH=10 pyrrolidine-acetic acid buffer solution. MP B, Methanol (containing 0.05 mol/L
of pH=10 pyrrolidine-acetic acid buffer salt). MP C, acetone (containing 0.05 mol/L of
pH=10 pyrrolidine-acetic acid buffer salt).
1.3 Standard Procedure
0-15 ml of 0.2 mg/ml standard or sample solution was transferred into a 25 ml
volumetric flask, for which, 5.0 ml of 1.0 ¡Á10-4 mol/L T2APP
THF solution, 2 ml of Triton X-100 solution and 3 ml of 0.5 mol/L pyrrolidine-acetic acid
buffer solution (pH=10) were added. The solution was diluted to the volume with water and
mixed well. The mixture was heated in a boiling water bath for 15 min. After cooling, the
solution was diluted to the volume with THF for subsequent analysis. 5.0 ml of solution
was introduced into the injector and sent to the enrichment column with mobile phase A at
a flow rate of 2.0 ml/min. After the enrichment had been finished, by switching the valve
of the six-port switching valve, the metal-T2APP chelates absorbed onto the
foreside of the enrichment column, were eluted by mobile phase B and C at the flow rate of
1.0 ml/min in reverse direction and traveled towards the analytical column and separated
on it. A tridimensional (X axis: retention time, Y axis: wavelength, Z axis: absorbance)
chromatogram was recorded from 350- 600 nm with a photodiode array detector and the
chromatogram of 450 nm is shown in Fig 2. In the course of separation, the composition of
mobile phase is: 0 min (100% B and 0£¥ C)£¬10 min (90 % B and 10£¥ C) in
linear ramp. Each metal-T2APP chelate was detected at its maximum absorption
wavelength.
Fig.2 Chromatogram of standard sample and water sample£ºa) Standard sample£¬b) Water
samples
Injection volume 5ml. The concentration of Ni(II), Hg(II), Pb(II), Cd(II) is 10 m g/L in
standard sample. Detection wavelength is 450 nm. Other conditions as in standard procedure
2. RESULTS AND DISCUSSION
2.1 Precolumn Derivation
In a weak alkaline medium of pH 8.5-12, Ni(II), Hg(II), Pb(II) and Cd(II) can form stable
and colored chelates with T2APP, so a 0.5 mol/L of pH=10 pyrrolidine-acetic
acid buffer solution was recommended to control pH.
It was found that
0.5 ml of 1.0¡Á10-4 mol/L T2APP THF solution was sufficient to complex 3.0 m g
of Ni(II), Hg(II), Pb(II) and Cd(II), respectively. But in real samples, the foreign ions,
such as Mg2+, Cu2+, Pd2+, Ru2+, Bi3+,
Co2+, Fe3+, Mn2+, Sn(IV), Bi3+, Zn2+
and the like, form complexes with T2APP and consume reagents. So the amount of
T2APP must be in excess. In this experiment, A 5.0 ml of 1.0¡Á10-4 mol/L
T2APP solution was recommended.
The reaction of Ni(II), Hg(II), Pb(II) and Cd(II) with T2APP
was slow at room temperature. Heating can accelerate the reaction. The reaction was
completed by heating in a boiling water bath for 15 min and the complex was stable for at
least 4 h after cooling, so that heating 15 min in a boiling water bath was selected.
2.2 On-Line Enrichment
Ni-T2APP, Hg-T2APP, Pb-T2APP and Cd-T2APP
chelates are stable in weak alkaline medium. To avoid the chelates decomposing during the
elution, a 0.05 mol/L of pH=10 pyrrolidine-acetic acid buffer solution (mobile phase A)
was selected as mobile phase to send the chelates to the enrichment column and a Waters
XterraTM RP18 chromatographic column (5mm, 3.9¡Á20 mm) with pH range
1- 12 was selected as enrichment column. Experiments showed that the volume of a 5 ml
sample injected was sensitive enough to determine Ni(II), Hg(II), Pb(II) and Cd(II) in
samples, so a 5 ml sample injection was recommended.
2.3 Spectrophotometric Properties
From a tridimensional chromatogram recorded by photodiode array detector, the
absorption spectrum of metal-T2APP chelates was obtained. The maximum
absorption wavelengths of Ni-T2APP, Hg-T2APP, Pb-T2APP
and Cd-T2APP are 434 nm, 452 nm, 468 nm and 440 nm, respectively. To get
maximum sensitivity, each metal-T2APP chelate was monitored at its maximum
absorption wavelength.
Fig.3 The effect of mobile phase pH on peak area
2.4 Chromatographic Separation
The Ni-T2APP, Hg-T2APP, Pb-T2APP and Cd-T2APP
chelates were stable in weak alkaline medium. So the effect of mobile phase pH on
chromatographic peak was studied (Fig 3). Experiments showed that the pH of mobile phase
within 8.8- 11.5 can avoid the chelates decomposing and get a maximum and constant peak
area. So two weak alkaline solutions, mobile phase B: methanol (containing 0.05 mol/L of
pyrrolidine-acetic acid buffer salt (pH=10.0)), and mobile phase C: acetone (containing
0.05 mol/L of pyrrolidine-acetic acid buffer salt (pH=10.0)) were recommended. Since the
common reserved phase chromatographic column could not remain stable at pH 10, a Waters
XterraTM RP18 chromatographic column (5 mm, 3.9¡Á150) was selected as analytical column in this experiment. XterraTM
RP18 columns have good stability in pH 1- 12. The relative proportions of
mobile phase B and C were varied to effect the separation. Experiment showed that gradient
elution achieved good results. The proper composition of mobile phase during the gradient
elution was selected as follows: 0 min (100% of B and 0£¥of C)£¬10 min (90% of B and 10£¥
of C) in linear ramp.
2.5 Calibration Graphs
Under optimum conditions, regression equations of
metal-T2APP chelates were established based on the standard samples injected
and their peak areas. Limits of detection are calculated by the ratio of signal to noise
(S/N=3). The reproducibility of this method was also examined for 10 mg/L of
Ni(II), Pb(II), Cd(II) and Hg(II). The results are shown in Table 1.
Table1 Regression Equation, Coefficient and Detection Limit
Components |
Regression
Equation |
Linearity
Range(mg/L) |
Coefficient |
Detect
limit (ng/L) |
RSD%(n=10) |
Ni-T2APP |
A=1.75¡Á106
C+122 |
0.01~ 120 |
r=0.9992 |
3.5 |
2.1 |
Cd-T2APP |
A=2.83¡Á106
C+135 |
0.01~ 120 |
r=0.9991 |
2.5 |
2.3 |
Pb-T2APP |
A=1.74¡Á106
C+212 |
0.01~ 120 |
r=0.9995 |
3.0 |
2.2 |
Hg-T2APP |
A=1.86¡Á106
C£208 |
0.01~ 120 |
r=0.9994 |
3.0 |
2.4 |
2.6 Interference
Under the pre-column derivatization condition, the foreign
ions of Mg2+£¬Cu2+£¬Pd2+£¬Bi3+£¬Co2+£¬Fe3+£¬Mn2+£¬Sn(IV), Zn2+£¬Pt2+, Ba2+ and Ag+ react
with T2APP to form color chelates. To examine the selectivity of this method,
the interference of foreign ions was investigated. When 5.0 ml of 1.0¡Á10-4 mol/L
T2APP was used, with 10 m g/L of Ni(II), Pb(II), Cd(II) and Hg(II),
respectively, the amount tolerated with an error of ¡À 5% is shown in Table 2. The results
showed that most foreign ions do not interfere with the determination. This method is
highly selective.
Table 2 Tolerance
amount of foreign ions (error of ¡À 5%)
¡¡ |
Tolerance
amount(mg/L) of foreign ion for 10mg/L of Sn(IV), Ni(II), Cd(II), Pb(II) and Hg(II) |
Ag(I) |
Mg2+ |
Cu2+ |
Pd2+ |
Bi3+ |
Co2+ |
Fe3+ |
Mn2+ |
Zn2+ |
Pt2+ |
Ba2+ |
Ni(II) |
500 |
25000 |
3000 |
1000 |
1500 |
1000 |
3000 |
3500 |
1500 |
1000 |
1000 |
Cd(II) |
1500 |
10000 |
2500 |
500 |
1000 |
1500 |
2500 |
4000 |
2000 |
2000 |
2000 |
Pb(II) |
1000 |
15000 |
1000 |
800 |
1500 |
1500 |
1500 |
5000 |
1500 |
500 |
2000 |
Hg(II) |
500 |
10000 |
1500 |
1000 |
1000 |
1000 |
3000 |
3000 |
1000 |
1000 |
1500 |
2.7 Application to Water Sample
For the fresh water (tap water, river water and lake water)
the water sample was analyzed according to the general procedure. The results (deducted
the reagents blank) were shown in Table 3, together with the results of a recovery test by
added 0.2 mg of Ni, Pb, Cd and Hg in water sample and diluted to 50 ml of final solution.
For plant effluents, the sample was digested as in the literature [6] and
analyzed according to the general procedure. The results (deducted the reagents blank)
were shown in Table 3 too, together with the results of a recovery test by added 0.2 mg of Ni, Pb, Cd
and Hg in water sample and diluted to 50 ml of final solution. A standard method using
atomic absorption spectrometry had also been used as reference method. The results are
shown in Table 4.
Table 3 Determination
results (mg/L) of the water
sample with this method
Components |
Samples (mg/L) |
RSD£¥(n=5) |
Recovery£¥(n=5) |
River water |
Lake
water |
Plant
effluent |
Tap
water |
Ni |
26.2 |
15.8 |
44.2 |
16.2 |
2.3 |
97-103 |
Cd |
5.46 |
11.2 |
16.7 |
2.22 |
2.2 |
97-104 |
Pb |
12.5 |
8.65 |
22.8 |
6.58 |
2.1 |
96-102 |
Hg |
2.87 |
5.62 |
12.5 |
- |
1.7 |
98-105 |
Table 4 Determination
results (m g/L) of the water sample with reference method
Components |
Samples (mg/L) |
RSD£¥(n=5) |
Recovery£¥(n=5) |
River water |
Lake
water |
Plant
effluent |
Tap
water |
Ni |
28.2 |
16.5 |
48.5 |
15.2 |
3.2 |
95-105 |
Cd |
5.96 |
11.8 |
15.2 |
2.13 |
3.5 |
96-108 |
Pb |
11.8 |
8.76 |
24.4 |
6.76 |
3.0 |
91-102 |
Hg |
2.81 |
5.41 |
13.5 |
- |
3.2 |
95-106 |
3. CONCLUSION
The proposed method has the following
advantages: (1) Four toxic heavy metal ions, Ni(II), Pb(II), Cd(II) and Hg(II) were
successfully separated in the proposed method using a buffer solution of pH 10 as mobile
phase on Waters XterraTM RP18 Column. (2) By an on-line enrichment
system, a large volume of sample (5 ml) can be injected. (3) With a photodiode array
detector, each metal-chelate can be monitored at its maximum absorption wavelength to
achieve maximum sensitivity. In a word, for the determination of nickel, lead, cadmium and
mercury ions in water, this method is highly sensitive and highly selective.
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