High-pressure Liquid-Liquid Partition Chromatography of Metal Chelates of Tetradentate ,&Ketoamines Enrico Gaetani and Carlo F. Laureri lstituto di Chimica Farmaceutica e Tossicologica, Universita di Parma, 43 100 Parma. Italy
Alessandro Mangia' and Giovanni Parolari lstituto di Chimica Generals ed Inorganica, Universita di Parma, 43 100 Parma, Italy
High pressure liquid-liquid partition chromatography was applied to some metal chelates of the tetradentate p-ketoamines: N,N' -ethylenebls(acetylacitonelmine), (H2(en)AA), N,N'-trimethylenebls( acetylacetoneimine), (H2(tm)AA) and N,N'-ethylenebb( benzoylacetonelmlne) (H2(en)BA). The complexes Coli, Ni", Cull, Pd"(en)AA, NI", Cu"(en)BA, Cu"(tm)AA have been consldered. Nickel and copper were separated by using H2(en)AA and H2(en)BA on two different columns. Palladiumwas successfully separated from copper but not from nickel. The dependence of the response of the wv detector (254 nm) on the amount of metal In aqueous solutions is reported for Ni and Cu(en)AA. The detection limits are about 0.2 and 0.5 ng of metal injected for NI and Cu, respectlvely.
High-pressure liquid chromatography is most widely employed in organic chemistry, but some examples of its application to inorganic and organometallic compounds have been reported. Ion exchange technique has been used for separation and determination of FeI" ( I ) , SbIII, BPI, Crvl, A@, HgII, PdI1, PtIV,RuIV,TlIII, SnIV(2), PblI ( 3 ) ,ThIV,Ca, CuII, Mn", Nil1 (41, rare earths (5), and transplutonium elements using di(2-ethylhexyl)orthophosphoric acid as a stationary phase (6). With liquid-solid high-pressure chromatography, the separation of HgII, CUI' (7), SnlI (8)has been achieved. The partition liquid-liquid technique, with direct or reversed phase, has been chosen for organometallic CrlI1 (9, 10) and FelI1 (11) compounds and for the P-diketonates of several divalent and trivalent metals (12). We applied the reversed phase liquid-liquid partition chromatography to some metal complexes of the tetradentate B-ketoamines
of CoII(en)AA, NilI(en)AA, Cdl(en)AA, PdlI(en)AA, Ni'I(en)BA, CuYen)BA, Cu"(tm)AA. The dependence of tlie detector response on the amount of Ni and Cu in aqueous solutions is also reported. EXPERIMENTAL Preparation of Ligands and Metal Chelates. Described procedures were followed in the preparation of Hz(en)AA, Hz(tm)AA, Hz(en)BA, Ni(en)AA, Cu(en)AA, Cu(tm)AA, Cu(en)BA ( 1 5 ) and Pd (en)AA( 16). Except for Hz(tm)AA,the compounds were characterized by elemental analysis, mp, and mass spectra. The mass spectra of the complexes of Hz(en)AAand Hz(tm)AAagree with those reported (17). The mass spectra of Ni(en)BA and Cu(en)BA show the expected fragmentation patterns with parent ion peaks. The cobalt(I1) complex was not isolated, but prepared by adding small excess of cobalt acetate to the methanolic solution of Hz(en)AA in nitrogen atmosphere. The yellow solution obtained was directly used for the chromatographic analysis. High-pressure Liquid Chromatography. Apparatus. A Varian Aerograph 8500,with a single wavelength uv detector (254nm) was used; the full-scale sensitivity was 0.005 absorbance unit; flow cell volume, 8 ~ 1 . Columns. The columns used were stainless steel 25 cm X 0.2cm i.d. MicroPak CH (Octadecylsilaneon silica gel 10-kmdiameter), stainless steel 50 cm X 0.2cm i.d. slurry packed (18) with silica gel (10-fimdiameter) bonded to 3-aminopropyltriethoxysilane(19) (-NH2 column). M phosphate or borate buffer As moving phase, methanol-6 X mixtures were used. The pH of the buffer solutions ranged between 7.0 and 11.0.The flow rate of the eluent was 1or 1.5 cm3 min-l at a pressure of 225-300 atm. Spectroscopicgrade solvents were used. Peak areas were measured by a Varian CDS 101 integrator. Mass Spectrometry. Mass spectra of ligands and chelates were run on a Varian MAT CH5 spectrometer at the ionizing voltage of 70 eV. The solid samples were directly introduced into the source by means of a direct insertion probe. Source temperature was 220-240 "C.
Ultraviolet-Visible Spectrometry. Electronic spectra of the chelates were run on a Perkin-Elmer model 402 spectrophotometer. Methanol and tetrahydrofuran (spectroscopic grade) were used as solvents. HC C ' -OH
/
R
with B = (CH,), B = (CH,), B = (CH,),
HO-C
/;""
RESULTS AND DISCUSSION N,N'-Ethylenebis(acety1acetoneimine)and its analogues,
\
R R = CH3 (H,(en)AA) R =CHj (H,(tm)AA) R = CeH5(Hden)BA)
Our aim was to study the behavior of metal chelates in high pressure partition chromatography and to evaluate the possibility of using this method in the determination of metals, The separation of Ni and Cu using the ligand Hn(en)AAhas been briefly reported by us (13). Independently and at the same time, other authors published the separation of Ni(en)AA and Cu(en)AA (14);in this case liquid-solid chromatography was used, with microparticulate silica as a stationary phase. The present paper deals with the chromatographic behavior
particularly the fluorinate ones, have been successfully employed in the gas chromatographic analysis (17,20-22).Owing to the high values of the stability constants of their complexes (log K = 23 for Cu(en)AA (23)),which are soluble in medium and low polarity solvents, these ligands are also suitable for liquid-liquid partition Chromatography; at high pH, the values of the total distribution coefficient of the metals are approximately coincident with those of the partition coefficient of the complexes (12). Moreover the high values of the molar absorptivities in the uv region allow good sensitivity with photometric detectors. The low number of metals with which they form stable complexes ( 2 4 ) ,reduces the problem of interferences. On the MicroPak CH column different moving phases were used, varying the volume ratio between methanol and the
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H2enAA
Co e n A A
i CuenAA
NI e n A A
a
b
C
Figure 1. (a)Separatlon of Ha(en)AA,Co"(en)AA,Ni"(en)AA,Cu"(en)AA.Column: MlcroPak CH 25 cm X 0.2 cm; moving phase: methanoVphosphate buffer pH 7.8, 65/35 (v/v); flow rate: 1 cm3 min-l. Theoretical plate number for Cu(en)AA: 480. (b) Superimposed chromatograms of Nl(en)AA and Pd(en)AA.Moving phase: methanol/phosphate buffer pH 7.8, 45/55 (v/v); flow rate: 1 cm3 min-'. (c) Separation: of Pd(en)AA and Cu(en)AA at the same condition of b). Theoretical plate number for Cu(en)AA: 410
Flgure 2. Separation of Cu(en)AA,Cu(tm)AA, and Cu(en)BA.Column: MicroPak CH; mobile phase: methanol/phosphate buffer pH 7.8,50/50 (v/v). Flow rate: 1 cm3 min-'. Theoretical plate number for Cu(en)BA:
4
940
b
aqueous buffers and the pH value of the buffer. Separation of a methanol solution of Hz(en)AA, Co, Ni, Cu(en)AA was achieved by using a methanolhuffer volume ratio 65/35 and pH 7.8 (Figure la). The resolution factors are 1.44 for Cu/ Ni(en)AA, 2.85 for Ni/Co(en)AA, 4.25 for Cu/Co(en)AA. The peaks were identified by injecting the single chelates. The cobalt chelate decomposes in solution, even in Nz atmosphere; the solution turns brown, and successive injections show a second peak immediately after that attributed to Co(en) AA. Separation of Ni(en)AA and Pd(en)AA was not successful, Pd(en)AA being only slightly more retained (Figure l b ) for every volume ratio between methanol and buffer solution. The separation of Pd(en)AA and Cu(en)AA in methanolic solution is shown in Figure IC.The resolution factor for Cu/Pd(en)AA under the conditions quoted in the figure, is 1.94. The behavior of CuenAA is not considerably influenced by the pH of the buffer on the range 7.0-11.0(phosphate buffers 1726
0
6
8
bmin
Figure 3. (a)Superimposed chromatograms of Cu(en)BAand Ni(en)BA. Column: MlcroPak CH; mobile phase: methanoVphosphate buffer pH 7.0, 70/30 (v/v). Flow rate: 1.5 cm3 mln-l. (b) Separation of Cu(en)BA and Ni(en)BA.Column: -NH2 50 cm X 0.2 cm; mobile phase: methanoVphosphate buffer pH 7.8, 40160 (v/v). Flow rate: 1 cm3 mln-l. Theoretical plate number for Ni(en)BA: 346
up to 8.0 and borate buffers between 8.0 and 11.0 were used): in this pH range, the uncorrected retention time lengthens from 1.7 to 1.9 min for a methanolhuffer volume ratio 70/30; the area of the peak is constant for the same quantity of complex. The copper chelates Cu(en)AA, Cu(tm)AA, Cu(en)BA in T H F solution are separated using a 50/50 methanolhuffer pH 7.8mixture (Figure 2). The presence of the phenyl ring in Cu(en)BA determines a major affinity of the complex for the stationary phase and the retention time increases sensibly. With this ligand, nickel and copper are not separated; the superimposed chromatograms of Ni(en)BA and Cu(en)BA in chloroform solutions are shown in Figure 3a. On
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I I
250
Figure 4. Ultraviolet spectra of 5 X Ni(en)BA in tetrahydrofuran
4
300
nm
350
M solutions of Cu(en)BA and
the contrary, Ni(en)BA and Cu(en)BA are separated on the -NH2 column, using a mobile phase with a volume ratio methanolhuffer pH 7.8,40/60 (Figure 3b). Both these chelates are stable and have high absorption in the uv region (Figure 4). Their molar absorptivities at 254 nm (t 20 300 and 26 000 mol-l 1. cm-1 for Cu(en)BA and Ni(en)BA, respectively) could allow a good sensitivity in the chromatographic determination of traces of Ni and Cu. The other complexes Ni, Cu, Pd(en)AA and Cu(tm)AA are not separated on the -NH2 column for any composition of the mobile phase; their retention times are influenced very little by the volume ratio in the eluent mixture. To estimate the possibility of an analytical application of the liquid-liquid partition chromatography of tetradentate ketoamines, the dependence of the detector response on the amount of the metal was verified for Ni(en)AA and Cu(en)AA. For this purpose, the MicroPak CH column was used; the mobile phase was a methanolhorate buffer p H 10, 7i)/30 mixture. Calibration curves were set up both with solutions of the pure chelates and starting from the metals in aqueous solution. In the latter procedure, methanolic solutions of an excess of the ligand (500-50 times) were added to equal volumes of aqueous solutions of the metal acetates in the range of concentration 0.2-2 and 10-100 ppm of metal, buffered at pH 10. The reaction was carried out three times for each sample; the mixed solutions were directly injected into the column. The injections were replicated five times and the peak area was determined by means of an integrator. Figure 5 shows a typical chromatogram of some injections of 5 pl of a 0.5-ppm solution of Ni2+, i.e., corresponding to 2.5 ng of metal. The calibration curves obtained starting from the aqueous solutions of the metals are linear in both ranges of concentration, corresponding to 0.5-5 and 25-250 ng of metal injected; the relative standard deviation of each mean value of the peak area is about 2%in the higher range of concentration and between 3-6% in the lower one. The plots obtained by using methanolic solutions of the two chelates coincide with those obtained starting from the metals, within the experimental errors. The detection limits, corresponding to a signal-to-noise ratio 2:1, for Ni and Cu are about 0.2 and 0.5 ng. The lower detection limit for Ni is attributable to the higher molar absortivity at 254 nm of the nickel chelate (13) and especially to its lower retention time. Moreover, it is noticed that the metals can be extracted by means of a chloroform solution of the ligand and the extracts can be concentrated; in this way, it is possible to start from more dilute aqueous metal solutions.
-0
2
0
,
, I , ,
2
0
2
, I / , 2
0
, ' ,
,
0
2
.
Figure 5. Chromatograms of successive injections of Ni(en)AA: methanol/water solutlon of 0.5 ppm of metal; 0.5-pl samples. Column:. MicroPak CH; mobile phase: methanoVborate buffer pH 10,70/30. Flow rate: 1.5 cm3 mln-'
The results obtained confirm the possibility of a useful application of the HPLC to metal chelates; further studies on liquid-liquid partition chromatography of the metal complexes of P-ketoamines and other polydentate ligands are in progress. LITERATURE CITED (1) M. D. Seymour, J. P. Sickafoose, and J. S. Fritz, Anal. Chem., 43, 1734-1737 11971). (2) M. D. Seymour and J. S. Fritz, Anal. Chem., 45, 1394-1399 (1973). (3) M. D. Seymour and J. S. Fritz, Anal. Chem., 45, 1632-1636 (1973). (4) J. S. Fritz, Anal. Chem., 48, 825-829 (1974). (5) D. 0.Campbell, J. lnorg. Nucl. Chem., 35, 3911-3919 (1973). (6) E. P. Horwitz and C. A. A. Bioomquist, J. lnorg. Nucl. Chem., 35,271-264 (1973). (7) P. Heizmann and K. Balischmiter. Fresenius 2. Anal. Chem., 288 (3). 206-207 (1973). ( 8 ) J. S. Fritz and L. Goodkin, Anal. Chem., 48, 959-962 (1974). (9) J. M. Greenwood, H. Veening, and B. R. Willeford, J. Organomet.Chem., 38,345-348 (1972). (IO) J. F. K. Huber, J. C. Kraak, and H. Veening, Chem. Commun., 1305 (1969). (11) R. E. Graf and C. P. Lillya, J. Organornet.Chem., 47,413-416 (1973). (12) J. F. K. Huber, J. C. Kraak, and H. Veening. Anal. Chem., 44,1554-1559 (1972). (13) E. Gaetani, C. F. Laureri, and A. Mangia, Abstracts, 12th National Meeting of the Societi Chimica itaiiana, Cagliari, September 1975, pp 66-70. (14) P. C. Uden and F. H. Walters, Anal. Chim. Acta, 78, 175-183 (1975). (15) P. J. McCarthy, R. J. Hovey, K. Ueno, and A. E. Martell, J. Am. Chem. SOC., 77,5820-5824 (1955). (16) "Gmelins Handbuch der Anorganische Chemie". 65, Verlag Chemie OBBH, Weinheim, 1942, p 403. (17) R. Belcher, K. Blessel, I.Cardwell, M. Pravica, W. I. Stephen, and P. C. Uden, J. lnorg. Nucl. Chem., 35, 1127-1 144 (1973). (18) R. E. Majors, Anal. Chem., 44, 1722-1726 (1972). (19) R. E. Majors and M. i. Hopper, J. Chromatogr.Sci., 12, 767 (1974). (20) R. Beicher, M. Pravica, W. i. Stephen, and P. C. Uden, Chem. Commun., 41-42 (1971). (21) P. C. Uden and K. Blessel, lnorg. Chem., 12,352-356 (1973). (22) R. Belcher, R. J. Martin, W. I. Stephen, D. E. Henderson, A. Kamalizad, and P. C. Uden, Anal. Chem., 45, 1197-1203(1973). (23) G. Schwarzenbach and M. Honda, Helv. Chlm. Acta. 40, 27-40 (1957). (24) R. H. Holm, G. W. Everett, Jr., and A. Chakravorty, "Progress in Inorganic Chemistry", Voi. 7, F. A. Cotton. Ed., Interscience. New York, 1968, p 180.
RECEIVEDfor review April 15,1976. Accepted June 14,1976. A part of this work was presented at the XI1 National Meeting of the Societl Chimica Italiana, Cagliari, Italy, September 1975.
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