Anal. Chem. 1988, 60, 2244-2248
2244
Detection of Metal Ions by Liquid Chromatographic Separation of Their 1,3-Dimethyl-4-acetyl-2-pyrazolin-5-one Chelates Margo D. Palmieri' a n d James S. Fritz*
Ames Laboratory and Department of Chemistry, Iowa State University, Ames, Iowa 50011
Metal ions are determined by adding 1,3-dimethyi-4-acetyl2-pyrazoiin-5one (DMAP) to an aqueous sample and then separating the metal chelates by dlrect InJectlononto a llquid chromatographic column. Separations on a C-18 slilca column and a polystyrene-dlvlnylbenzene column are compared, with a better separation seen on the polymeric column. I t Is found that the moat emcienl separations are seen at pH 5 with an acetk acid buffer. Separatlons of metai-DMAP complexes using dtffereni organlc modiflers are compared. Good separations uslng acetonitrile and tetrahydrofuran are seen, and an inversion of retention Is seen in tetrahydrofuran. Several metal ions can be separated and quantified; separatlon conditions, llnear callbration curve ranges, and detection limits are presented for Fe(III), U(VI), Ga(III), and Cu(I1). Interferences due to the presence of other ions in solution are investlgated. Finally a method for selectively determlning uranium with postcolumn reactor detection using Arsenazo I Is presented.
An attractive method for determining metal ions solution is to form a metal complex with an organic chelating agent and then separate the complexes by high-performance liquid chromatography (HPLC). The extensive literature on this type of separation has been covered in several reviews (1-6). In general, it has only been possible to separate a limited number of metal complexes in a single chromatographic determination. Metal complexes often tend to dissociate or decompose during passage through the chromatographic system. The labile nature or weak formation of many metal complexes, the presence of metal parts in some chromatographic systems, and ion-exchangeproperties of silica supports are all responsible for the small number of metal complexes separated. In a previous paper by the present authors, excellent separations of several 3+ and 4+ metal-hydroxamic acid complexes were achieved by using a polymeric resin and eluents containing hydroxamic acid (7).The polymeric organic resin was used to avoid the ion exchange properties associated with silica-based materials. The addition of hydroxamic acid to the eluent prevented dissociation of the labile metal complexes and reduced the effects of metal chromatographic parts on the separation. Several authors have used /3-diketones for complexation of metal ions prior to chromatographic separation (8-14).In the present work, the compound 1,3-dimethyl-4-acety1-2pyrazolin-5-one (DMAP) is used to complex the metal ions of interest. DMAP belongs to a particular group of P-diketones called I-acyl-2-pyrazolin-5-ones; it has been synthesized and characterized by King (15). Both DMAP and its metal complexes are fairly soluble in water, yet the metal complexes are readily retained on a hydrophobic resin column. One other study has been carried out using a 4-acyl-2pyrazolin-5-one as a complexing agent for the separation of trace metals by HPLC. Morales and Bartholdi separated Present address: National Bureau of Standards, Gaithersburg,
MD 20899.
0003-2700/88/0360-2244$01.50/0
uranium(VI), iron(III), thorium(IV), copper(II), zirconium(IV), and neptunium(1V) complexes of l-phenyl-3-methyl-4benzoyl-2-pyrazolin-5-one(PMBP) by using a (2-18 column and an acetonitrile-water eluent (16). A high percentage of organic modifier was required (>90%) because the complexes are very hydrophobic and are insoluble in water. They found the addition of a small amount of PMBP to the eluent improved peak shape of the complexes. EXPERIMENTAL SECTION Synthesis of DMAP and Preparation of Solutions. 1,3Dimethyl-2-pyrazolin-5-one was synthesized from methylhydrazine and ethylacetoacetate and then reacted with acetyl chloride to form DMAF', according to the procedures described by King (15). (Caution: methylhydrazine is a suspected carcinogen). AU organic reagents were purchased from Aldrich. Solutions of DMAP were prepared by dissolving DMAP in water. Metal ion stock solutions were made from metal ion salta or from the metal. The salts and metals were obtained from various sources. Uranium(VI), copper(II), thorium(IV), and iron(II1) solutions were made from the nitrate salta. Vanadium(lV)solution was prepared by dissolving vanadyl sulfate. Aluminum and gallium metals were dissolved in hydrochloricacid and nitric acid solutions, respectively. Stock solutions used in the interference studies were made from nitrate, perchlorate, chloride, and sodium salta. The DMAP metal complexeswere prepared by adding an excess of DMAP and buffer to a metal ion solution. Spectral studies were carried out on these solutions by using a DMAP blank. LC Studies. The chromatographic system consisted of a Milton Roy simplex minipump, Milton Roy pulse dampener, pressure release valve, 10 cm X 4 cm saturator column, Rheodyne 7010 injector with a 20-pL sample loop, 0.2-pm Rheodyne in-line Titer, and a Tracor 560A UV-vis scanning detector. The saturator column was hand packed with silica gel (Amico)or XAD 16 ( R o b and Haas). Two types of columnswere used: a 3-pm Zorbax C-18 silica column (40 mm X 6 mm) from Du Pont and a PLRP-S 5-pm poly(styrene-divinylbenzene) (150 mm X 4.6 mm) column (PS-DVB) from Polymer Laboratories. Eluents were prepared from Fisher HPLC grade acetonitrile, tetrahydrofuran, or methanol and from water purified with a Barnstead Nanopure I1 system. Acids and buffers were reagent grade or better. Eluent components were mixed together and filtered with a 0.2- or 0.45-pm Nylon 66 (Rainin) or PTFE filters (Nuclepore). Eluents were made fresh every day. For all eluents except those containing tetrahydrofuran, the flow rate was set at 1.0 mL/min. When tetrahydrofuran was the organic modifier, the flow rate was reduced to 0.6 mL/min because of the higher back pressure. For direct detection the detector wavelength was set at 318 nm. The eluents used for the majority of the studies contained 5 X lo4 M DMAP and either 0.02 M pyridine or acetate (HAC)buffer. Apparent pH values of the eluent were measured with a Corning 125 pH meter and adjusted by using nitric acid or NaOH solutions. All pH values reported are apparent pH because the eluent contained an organic solvent in addition to water. For the postcolumn reactor studies, an LKB 2150 HPLC pump delivered a postcolumn reagent solution of 2.5 X lo4 M Araenazo I (Kodak)at approximatelythe same flow rate as the eluent. An organic modifier was added to the postcolumn reagent solution to prevent formation of air bubbles when the reagent and eluent were mixed together. The solution contained 0.2 M triethanolamine(TEA)-nitric acid buffer, adjusted to pH 7.8. A standard low-pressuretee was used. After the eluent and the postcolumn 0 1988 American Chemical Society
2245
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
reagent were mixed, the pH of the resulting mixture was 7.6.
RESULTS AND DISCUSSION Conditions for Separation. Detection Wavelength. Absorption spectra of several metal-DMAP complexes showed absorbance between 300 and 345 nm. Most had molar absorptivities of 3000 or less. By measurement of chromatographic peak heights and noise at several wavelengths, a wavelength of 318 nm was chosen for detection of the chromatographic peaks. Eluent Composition and p H . From earlier work on the adsorption of metal-DMAP complexes by XAD-4 resin (15), an initial pH of 5.0 was chosen for the eluent buffer. Initial chromatographictesting was carried out by using the PLRP-S column, a poly(styrene-divinylbenzene) gel, because of its similarity to XAD resins and because it contained no reactive sites such as found in silica-based columns. Initially, acetonitrilewater eluents containing no DMAP were used, but no metal-DMAP peaks were observed. Upon addition of DMAP to the eluent, metal-complex peaks were seen. Other groups have reported that the addition of complexing agent to the eluent improved peak shape (7,8,16). Varying the concentration of DMAP in the eluent did not affect the peak retention times, but the peaks were broader at lower DMAP concentrations. No thorium peak was observed below 0.1 mM DMAP. An eluent concentration of 0.5 mM DMAP was found to give satisfactory peak shape for most of the metal complexes tested. Figure 1 shows a chromatogram for a mixture of six metal-DMAP complexes obtained by using a pH 5.0 acetonitrilewater eluent. (Eluent conditions: 30% CH,CN, 70% HzO, 0.02 M HAC, 0.5 mM DMAP; flow rate, 1.0 mL/min; detection at 318 nm.) The copper(I1) and vanadium(IV)peaks are not resolved, but thorium(IV), uranium(VI), gallium(II1) are nicely separated. The early elution of the broad thorium peak is probably due to the hydrolysis of thorium. Morales and Bartholdi also reported hydrolysis of thorium PMBP complexes (16). Several other metal-DMAP peaks were also observed in other chromatograms. Aluminum(II1) eluted as a broad peak with essentially the same retention time as thorium(1V). Zirconium(n7) coeluted with uranium(VI) under these conditions. An unstable titanium(1V) peak was sometimes seen between thorium(1V) and uranium(V1). The pH of the eluent was varied to see if the separation could be improved or if additional metal-DMAP peaks could be seen. At pH values much above 5.0, direct detection was not possible because of the high absorbance of the anionic form of DMAP at 318 nm. The pK, for DMAP is 4.73 (15). As the pH was lowered, the metal complexes began to elute earlier. At pH 4.0, the thorium(1V) peak coeluted with copper(I1); gallium eluted approximately 2 min earlier and partially overlapped the uranium(V1) peak. Uranium(VI), zirconium(IV), and iron(II1) retention times remained approximately the same. Aluminum(II1) eluted as a broad, tailed peak at the same retention time as copper(I1)and thorium(IV). When the eluent pH was lowered to 3.0, the iron(II1) peak was not greatly affected, but the other peaks eluted much earlier. No peaks were seen for thorium or copper. Of the metal ions tested at pH 2.0, only uranium(V1) and iron(II1) peaks were seen. In the eluents of lower pH, the metal-DMAP complexes are not as stable, and the complexes dissociate as they travel through the column. From these studies it was concluded that the eluent pH of 5.0 is best for most separations. Different buffers were tested to see the effect of the buffer on the separation. Little difference in retention time or peak height was seen with pyridine, acetic acid, or hexamethylenetetramine at pH 5.0. Nicotinic acid adversely affected the quality of the gallium(II1) peak. The gallium peak
U
I W
VI
z
gVI W E
a
eY
Ga
IW
n
\
1 u 0 1
l
l
l
l
I
6 8 10 12 14 T I M E IN MINUTES
Figure 1. Separation of Cu-, V-, U0,-, plexes at pH 5.0 on a PLRP-S column.
1
1
1
16 16 2 0
Ga-,and Fe-DMAP com-
eluted earlier and the peak was smaller. Nicotinic acid probably forms a complex with gallium and competes with DMAP for gallium, which causes the poor retention characteristics of gallium. Eluent modifiers were also investigated briefly. Addition of sodium perchlorate caused thorium(1V) to elute as a shoulder on the front edge of the uranium(V1) peak. The increased retention time for thorium when sodium perchlorate was added indicated that thorium was probably a positively charged complex and supports the theory that the thorium complex hydrolyzes. Column. Because HPLC separations are most commonly carrier out on silica columns with a bonded organic phase, separations of metal-DMAP complexes were carried out on a C-18 silica column. The separation on the silica column is inferior to that on the polymeric column. Vanadium(IV), gallium(III), copper(II), and uranium(V1) all elute within 8 min, while the iron complex elutes as a broad peak at 20 min. The retention order is also different on the C-18 column. The gallium-DMAP complex elutes earlier than uranium(V1)and copper(I1)complexes, and it coelutes with the vanadium(1V) complex. No thorium(V1) peak is seen in the C-18 chromatogram. Interaction with the column silanol groups is probably breaking up the thorium(1V) complex. Other workers have reported interaction of silanol groups with metal complexes (17-20). mSiO-
+ Th(DMAP), e (-SiO-),Th4+ + nDMAP
(1)
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
2246
IO
05
cu
-9
.?
w
0
io
Z
0 LL
00
:I
-0 5
10
20
30
40
50
PERCENTAGE ACETONITRILE
Figure 2. Dependence of log k'on percentage acetonitrile concentration In the eluent for the PLRP-S column.
Eluent Solvent. The successful separations obtained on the polymeric PLRP-S column with acetonitrilewater eluents led to an investigation of the effect of eluent composition on retention. Figure 2 shows slightly curved plots of log 12 'against the percentage of acetonitrile in the eluent. Thus, relatively small changes in the proportion of acetonitrile in the eluent will give either earlier or later elution of the peaks. Methanol-water eluents (buffered and containing DMAP) were tried for separations on polymeric PLRP-S columns. Compared with acetonitrile, a much higher concentration of methanol was needed in the eluent for elution of the metal complex peaks. In order to separate vanadium(IV),copper(II), thorium(IV), uranium(VI), gallium(III), and iron(III), an eluent with an apparent pH of 5.0 containing 77% methanol, 23% water, and 0.5 mM DMAP was required. The elution order was vanadium(1V) < copper(I1) < thorium(IV), uranium(V1) < gallium(II1) < iron(II1). Copper(I1) was only partially resolved from the coeluting thorium(1V) and uranium(V1) complexes. The gallium(II1) and iron(II1) peaks eluted as broad peaks at 14 and 23 min, respectively. The high percentage of methanol required for this separation indicates that the resin is poorly wetted by methanol. Other researchers have reported the poor wetting ability of the PLRP-S column with methanol (21). Differences in solubility or possible adduct formation of the different DMAP complexes with methanol are probably responsible for the selectivity differences between the methanol and acetonitrile eluents. These results clearly show methanol-water eluents to be inferior to acetonitrile-water. Tetrahydrofuran (THF)-23% water eluents were also tested on the polymeric column. Excellent separationswere obtained (Figure 31, with the uranium-DMAP complex now eluting after the iron(II1) peak. (Eluent conditions: 23% THF, 77% H20, 0.02 M HAC, 0.5 mM DMAP; flow rate, 0.6 mL/min, detection at 318 nm.) The percentage of THF in the eluent
I
I
I
0
2
4
I
l
6 8
U
l
I
l
10 12 14 TIME 1N MINUTES
l
l
16 18
Effect of THF on the separation of Cu, Ga,Fe, and DMAP complexes at pH 5.0 on the PLRP-S column. Figure 3.
was approximately the same as that of acetonitrile in the eluent in Figure 1. The inversion of retention of the uranium-DMAP complex and iron-DMAP complex could be due to THF entering the coordination sphere of uranium, causing the complex to become more hydrophobic and thus increasing the retention. Researchers using THF as a solvent in combined ion exchangesolvent extraction reported greater complexation between THF and uranium (22) than with other solvents. @-Diketonecomplexes are also known to form adducts with various solvents (14). Sample Preparation. Before being chromatographed, an excess of DMAP and a buffer were added to the sample. This prederivatization ensured that the DMAP complex formed completely and prevented hydrolysis of the sample ions. Without prederivatization, the sample peaks were not as sharp and the various peaks often overlapped. Quantitative Results. The DMAP complexes were investigated to determine if the separation could be used as a quantitative method for determining metal ions. The mobile phase composition for each metal was adjusted so that the metal complex eluted at approximately 5 min. The 5-min elution was chosen because the peaks were sharp and narrow with little tailing and completely resolved from the void volume. Table I gives the eluent conditions, linear calibration curve range, and detection limits for copper(II), uranium(VI), gallium(III),and iron(II1). The detection limit was defined as the metal ion concentration, which gave a peak height 3
ANALYTICAL CHEMISTRY, VOL. 60, NO. 20, OCTOBER 15, 1988
Table I. Eluent Conditions for Analysis of Various Metals by HPLC Using DMAP as Complexing Agent linear calibration curve range, M
metal ion elution conditions Fe(II1)
40% CH3CN 60% Hz0 0.50 mM DMAP 0.02 M HAC
5
Cu(I1)
25% CH&N 75% HzO 0.50 mM DMAP 0.02 M HAC
7.8
Ga(II1)
35% CH&N 65% H,O 0.50 mM DMAP 0.02 M HAC
2
X
10*1.3
U(V1)
35% CHBCN 5 65% HzO 0.50 mM DMAP 0.02 M HAC
X
104-5
35% THF 65% H20 0.50 mM DMAP 0.02 M HAC
X
104-6
lo4
X
detection limit, M 2 x 10"
Table 11. Percentage Decrease in Peak Area of Gallium in 200-Fold Excess of Interferent When Preparing Samples at pH 3.0 and pH 5.0a Ionb
% decrease pH 5.0
% decrease pH 3.0
>99 28 12 99 9.5 12 4.1 61.5 3.5
7.4 8.4 e2 10 5.8 e2
La3+ NiZt
ZnZt Sm3+ X
104-3.1
X
X
X
5 x 104-1 x 10-5
6
X
10"
4
X
10"
5 x 10"
2247
COZ+
Mn2+ FHP04-2 PbZt "Eluent conditions: 30% CH,CN, 70% HzO, 0.02 M HAC,5 X lo4 M DMAP, pH 5.0; flow rate, 1.0 mL/min; 318 nm; [Gal = 1 X lo4 M, [interferent]= 2 X M. *Thefollowing ions tested gave less than 3% decrease in peak area for Ga when tested Kt, Na+, Ba2+,Ca2+,CdZt,NO3, C1-, ClO,, SO4". The concentrations of the ions were 2 X M.
2 x 10"
times larger than the background noise. Depending upon the metal ion, the calibration curves were linear for approximately 1 to 2 orders of magnitude. Peak area measurements of replicate injections were within 5% of each other. Uranium(VI) determination using tetrahydrofuran in the eluent had larger linear ranges and lower detection limits due to the increased absorption Qf the uranium-DMAP complex in tetrahydrofuran. No linear calibration curve for thorium(1V) was obtained. As the concentration of thorium(1V) decreased, the thorium(1V) peak broadened out and the peak area and height decreased nonlinearly. This behavior also indicates that the thorium is hydrolyzing in the chromatographic system. A 200-fold molar excess of the following ions gave no interference (
