178
Anal. Chem. 1982, 5 4 , 178-181
mixtures following silylation and use of a GC internal standard, ethylvanillin, that chemically resembles the predominant aldehydic oxidation products of lignin, both minimize artifacts due to carbonyl loss and allow excellent analytical precision for these compounds (Table 11). If necessary for particularly difficult samples, an additional noncarbonyl GC internal standard can be used so that the extent of aldehyde loss can be estimated from changes in its ratio to ethylvanillin. A third potential problem that is also quite variable in its impact is geometric isomerization of the cinnamyl phenols in silylated standard and sample solutions. The p-coumaric and ferulic acid produced by lignin oxidation and most commercially available counterparts are essentially pure trans geometric isomers. However, for some as yet unknown reason, slow isomerization to the corresponding cis form occurs for both compounds in some sample and standard solutions following silylation. Ferulic acid typically isomerizes much faster than p-coumaric acid. Geometric isomerization is too slow to have a measurable affect when GC analysis of sample oxidation product mixtures is performed immediately after silylation. However, isomerization effects can be significant (up to 10%/day) for some standard mixtures whereas others may be stable for weeks. Routine monitoring of silylated standard solutions for conversion of trans cinnamyl phenols to earlier eluting cis isomers (Figure 2a) is, therefore, prudent. The final problem that is sometimes encountered in the described analytical procedure is “superoxidation” of the sample as indicated by consistently high acid/aldehyde ratios within the p-hydroxyl, vanillyl, and syringyl phenol families (e.g., Table IV). These high ratios primarily reflect conversion of aldehydes to the corresponding carboxylic acids. Although such conversions in themselves have little effect on the lignin parameters P, V, and S or their derivative parameters (Table 11),other compositional artifacts such as preferential loss of syringyl phenols and overall decreased phenol yields also occur which are not corrected by summation within phenol families. Superoxidation can result from introduction of O2into the minibomb during charging or from elevated reaction tem-
peratures (20). The problem is most likely to occur in samples containing little organic matter.
ACKNOWLEDGMENT The authors thank P. Parker, T. C. Hoering, and R. Carpenter for numerous contributions. LITERATURE CITED (1) (2) (3) (4) (5) (6) (7)
(8) (9) (10) (11) (12) (13) (14) (15) (16) (17)
(18) (19) (20)
Sarkanen, K. V.; Ludwig, C. H. “Lignins”; Wiley: New York, 1971. Morrison, R. I. J. Soil Sci. 1963, 74, 201-216. Leo, R. F.; Barghoorn, E. S. Science 1970, 768, 582-584. Schnitzer, M.; Khan, S. U. “Humic Substances in the Environment”; Marcel Dekker: New York, 1972. Dormaar, J. F. Can. J. SoiiSci. 1979, 59, 27-35. Hedges, J. I.; Mann, D. C. Geochim. Cosmochim. Acta 1979, 43, 1809- 18 18. Pearl, 1. A. “The Chemistry of Lignin”; Marcel Dekker: New York, 1967. Chang, H.-M.; Aiian, G. 0.I n ”Lignins”; Sarkanen, K. V., Ludwig, C. H., Eds.; Wiley: New York, 1971; pp 433-485. Freudenberg, K.; Lautsch, W.; Engler, K. Chem. Ber. 1940, 73, 167. Farmer, V. C.; Morrison, R. I . Geochim. Cosmochim. Acta 1964, 28, 1537-1546. Gardner, W. S.; Menzel, D. W. Gsochim. Cosmochim. Acta 1974, 38, 8 13-822. Nelson, P. F.; Smith, J. 0.Tappi 1966, 4 9 , 215-217. Knapp, D. R. “Handbook of Analytical DerivatizationReactions”; Wiiey: New York, 1979. Hartiey, R. D. J. Chromatogr. 1971, 5 4 , 335-344. Schnitzer, M. SoiiBioi. Biochem. 1974, 6 , 1-6. Hayatsu, R.; Winans, R. E.; Scott, R. G.; McBeth, R. L.; Moore, L. P.; Studier, M. H. Science 1980, 207, 1202-1204. Hedges, J. I.; Parker, P. L. Geochim. Cosmochim. Acta 1976, 4 0 , 1019-1029. Ugolini, F. C.; Reanier, R. E.; Rau, G. H.; Hedges, J. I. Soil Sci. 1981, 737,359-374. Hedges, J. I.; Mann, D. C. Geochim. Cosmochim. Acta 1979, 4 3 , 1803-1 807. Hedges, J. 1. Ph.D. Dissertation, University of Texas at Austin, Austin, TX, 1975.
RECEIVED for review August 18, 1981. Accepted October 20, 1981. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. This research was also funded in part by NSF Grants OCE-7818260 and OCE-8023970. This is Contribution No. 1238 from the School of Oceanography, University of Washington, Seattle, WA.
Separation of Cationic Metal Chelates of 1 , lO-Phenanthroline by Liquid Chromatography Jerome W. O’Laughlln Department of Chemistry, Universi@ of Missouri, Columbia, Missouri 652 1 1
Ion palr, hlgh-performance llquld chromatographic separatlon of the Inert 1,lO-phenanthrollne chelates Fe(phen):+, Ru(phen)32+, and Nl(phen),’+ on polystyrene-dlvlnylbenzene polymer based columns Is reported by use of acetonltrllewater-perchlorlc acld mlxtures as the moblle phase. The extenslon of thls technique to the separation of the lablle chelates Zn( phen),”, Co( phen);’, Cd( phen),”, and Cu(phen),”, when the moblle phase Is l o 4 M In the ligand and LICIO, Is used In place of HCIO, as a source of the palrlng Ion, Is shown to be feaslble. The effects of moblle phase parameters on retention and resolutlon are reported on several dlfferent types of columns.
The separation of the 1,lO-phenanthroline chelates Ni0003-2700/82/0354-0178$01.25/0
(phen):+ and Ru(phen):+ from Fe(phen):+ but not from each other by ion-pair, high-performance liquid chromatography has been previously reported (1). The separation of all three of the above chelates from each other and the extension of this technique to the separation of the labile 1 , l O phenanthroline chelates of Zn(II), Cd(II), Co(II), and Cu(I1) on fi-Partisil-SCX(cation exchange) and the Hamilton-PRP-1 (polystyrene-divinylbenzene bead) columns with the perchlorate ion as the pairing ion is reported in the present paper. The effects of mobile phase parameters, volume percent acetonitrile, pH, the perchlorate ion concentration, and the 1,lO-phenanthroline concentration on the retention volumes are reported. The successful separation of the labile chelates when the mobile phase was kept 10”. M in l,l0-phenanthroline is shown. The retention volumes were found to decrease exponentially with an increase in the perchlorate ion con@ 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982
179
centration and the second power dependence observed is in agreement with equations developed by Eksborg and Schill (2) and Tomlinson and co-workers (3) if the mobile phase is regarded as the organic phase. Although an ion exchange column is used, it is shown that the distribution of the cationic chelates is not due to ion exchange but to the distribution of the chelate species paired with perchlorate ions between the mobile and stationary phases. The distribution process is fundamentally different than the ion exchange separation of the metal cations by high-performance liquid chromatography recently reported by Cassidy and Elchuck (4).
EXPERIMENTAL SECTION Chemicals and Reagents. The 1,lO-phenanthrolinemono-
hydrate was obtained from the Fischer Scientific Co. The acetonitrile used to prepare the mobile phases was "Omnisolv Grade Solvent" obtained from the Taylor Chemical Co., St. Louis, MO. The water employed was deionized water which was then distilled in glass. All other chemicals were reagent grade except the LiC104 which was prepared by treatment of ultrapure Li2C03 with perchloric acid and recrystallization of the LiC104 from water. The preparation of the solid tris(1,lO-phenanthroline)chelates as the iodide salta was described previously (1). Solutions of the labile metal chelates were prepared by dissolving appropriate amounts of the metal sulfates in water or the mobile phase and adding a small excess of 1,lO-phenanthroline(1). The solutions were then diluted to the appropriate volume as to be in the range of 1-100 ppm in the metal. Solutions of the complexes were also prepared by dissolving an appropriate amount of the solid complexes in acetonitrile (or the mobile phase). Instrumentation. The HPLC system used consisted of a Waters Associates 6000 A pump and U6K injector, and a Spectromonitor I, variable wavelength, photometric dectector with an 8-pL flow cell (Laboratory Data Control Co.). A 1-mVHoneywell Electronik-194 dual channel recorder was used to record the output. An Alltech Associates inlet fiilter (2 pm) was used between the injector and pump. The columns employed were a 10-pM Partisil-SCX and Partisil-SAX (4.6 mm i.d. and 25 cm in length) obtained from Whatman, a 10-pm Zorbax-CN column (4.6 mm i.d. and 25 cm in length) obtained from DuPont, and 10-pmPRP-1 column (4.1 mm i.d. and 15 cm in length) obtained from Hamilton. The mobile phases were prepared by volume. Acetonitrile and water solutions were prepared to have the same concentrations of Li(C104),HC104,and 1,lO-phenanthrolineand then appropriate volumes mixed. The mobile phase was purged with helium for several minutes before use. Samples containing from 5 ng to 5 pg of the various metals were injected with Hamilton microliter syringes and eluted under isocratic conditions. An attenuation setting of 0.32 was employed which corresponded to 1.26 X lo4 AU/mm for the 1mV recorder used.
J-7e-hMINUTES
Flgure 1. Separation of Ni(phen)t+, Ru(phen)l+, and Fe(phen);+ by HPLC: column, pPartlsil-SCX detection, 265 nm; mobile phase, 4 1 CH,CN-H,O, 0.06 M HCIO,; temperature 20 'C; flow rate, 1,OO mL/min; (1) Hphen', (2) Ru(phen):+, (3) Ni(phen);+, (4) Ru(phen)l+, (5) Fe(phen)t
.
1
40
Q:
L I
I
RESULTS AND DISCUSSION The separation of the inert chelates N i ( ~ h e n ) ~ Ru~+, hen)^^+, and F e ( ~ h e n )on ~ ~a+p-Partisil-SCX column with an 4:l acetonitrile-water mixture 0.06 M in perchloric acid as the mobile phase is shown in Figure 1. All three peaks are resolved and well separated from a peak due to the ligand, Hphen+, and a minor peak believed to be a bis(1,lOphenanthroline)ruthenium(II) species. This minor impurity was observed previously (1)and found to have essentially the same UV spectrum and fluorescence characteristics as the R ~ ( p h e n ) , ~species. + The retention of all these species was found to increase with the water content of the mobile phase as shown in Figure 2 for F e ( ~ h e n ) and ~ ~ +N i ( ~ h e n ) ~ Calculated ~+. values for the resolution of these two species at 90%, 80%, 70%, and 60% acetonitrile were R, = 1.47, R, = 1.55, R, = 1.20, and R, = 1.18, respectively,based on observed VR values and half-peak height measurements for a flow rate of 2 mL/min. The retention volumes for both the Ni(phen):+ and Fehen)^^+ species decreased exponentially with increasing perchloric acid concentration as shown in Figure 3. Excluding
I
eo
I
I
I
I
80
70
60
50
% C H3CN
Flgure 2. Variation in retention volume with percent acetonitrile: column, p-PartislCSCX mobile phase, CH,CN-H,O; detection, 265 nm; temperature = 23 'C; Fe(phen),*+ (0),Ni(phen),+ (A).
the datum point for 0.12 M HC104where VR was not much larger than VM, the data for Fe(phen):+ fit the linear equation log
V k = 1.984 log [C104-] - 1.3634
with a correlation coefficient of 0.9992. The corrected retention volumes, V $ = V R - VM, were calculated taking V M = 3.00 mL. The fact that the observed behavior was due to the perchlorate ion and not the hydronium ion was deduced in part from the fact that varying the [H30+]by the addition of phosphoric acid to the mobile phase had no significant effort
160
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2,FEBRUARY 1982
Table I. Retention Volume Data for Inert and Labile 1,lo-Phenanthroline Complexes on p-Partisil-SCXColumn mobile phase (23 "C) LiClO,, 1,lOCH3CN M phen,M 94.3 0.0238 10-4 94.3 10 -4 0.0691 90 10-4 0.0471 80 10-4 0.0710 80 10-4 0.047 70 10-4 0.047
VR, mLa
%
60 48 100 90 80 75 70
0.198 0.1 58 0.0566 0.0566 0.0566 0.0566 0.0566
10-4
phen
Co
Zn
Ni
Ru
54.5 5.82 5.41 5.31
16.1
16.5 8.85
16.5 8.85 16.2 26.8 6.53 17.7
11.2 17.8 9.60
Fe 62.1 12.0 19.5 10.0 18.8 33.8 7.10 21.o 7.5 13.2 13.6 16.7 23.3
Cd
cu
20.3 12.10 21.0 24.0
10-4 16.9 18.1 19.1 26.0 25.6 10-3 (monitored at 448 mm) 7.5 10-3 12.8 10-3 12.8 10-3 15.9 10-3 22.0 a Elution monitored at 265 nm except as indicated and in a number of cases Ru(phen)3z+ and Fe(phen),z+in mixtures were monitored at 448 and 512 nm as well as at 265 nm. on VR at various concentrations of the perchlorate ion when the mobile phase [C104-] was adjusted with lithium per180chlorate. At a lithium perchlorate concentration of 0.047 M for the case where the acetonitrile-water ratio was 4:1, re160tention volumes for Ni(phen)2+ and Fe(phen)t+ of 16.37 and 18.81 were observed with a calculated value for the resolution of R, = 1.55 (very similar to the retention behavior and res140olution observed for comparable concentrations of perchloric acid, Figure 3). The fact that retention of these cationic chelates was not 120due to ion exchange was confirmed by studying the retention of these species on a 15-cm PRP-1 column which is packed with 10-pm beads of a styrene-divinylbenzene copolymer. The 100retention volumes for Fe(~hen),~+, N i ( ~ h e n ) ~and ~ + ,RuP hen)^^+ were 6.88 mL, 7.72 mL, and 8.22 mL, respectively, 80on this column when a mobile phase 45:55 in acetonitrilewater and 0.03 M in perchloric acid was used. For a mobile phase 27:73 in acetonitrilewater and 0.02 M in perchloric acid, 60 V , values of 41.3 and 49.6 mL were obtained for F e ( ~ h e n ) ~ ~ + and Ni(phen)z+ with a plate count N = 1500 and a calculated value for the resolution, R,, of 1.75. As in the case of the 40Partisil-SCX column, the retention volumes decreased with increasing ratios of acetonitrile to water and perchlorate ion concentration. All three species were eluted from the PRP-1 20 column at the column volume of 1.96 mL with a mobile phase 80% in acetonitrile and 0.06 M in HC104. The variation in I I I I I I retention behavior with the mobile phase parameters studied .02 .04 .06 .08 .lo .I 2 was similar on the PRP-1 and Partisil SCX columns with the Flgure 3. Variation of retention volume with perchloric acid concenlatter showing somewhat greater retention. It should be noted, tration: column, p-Partisil-SCX; mobile phase, 4: 1 CH,CN-HpO; dehowever, that the elution order is different-Ni, Ru, and Fe tection, 265 nm; temperature 23 O C ; Fe(phen),*+(0),Ni(phen);+ (A). for the Partisil column and Fe, Ni, and Ru for the PRP-1 column. The elution order of the inert iron, nickel, and ruwas too large to permit UV detection at 265 nn but both the thenium complexes appears to be characteristic of the pariron and ruthenium species could be monitored at 448 nm. ticular column and does not change with composition of the It was possible to use UV detection if the ligand concentration mobile phase as previously suggested as a possibility (I) alM and in the mobile phase was kept to approximately though the retention volumes of the labile complexes do vary well-shaped peaks for Co(phen):+, Zn(~hen),~+, Cd(phen)?+, with ligand concentration. The mechanism by which metal and Cu(phen):+ were observed. Retention volumes for these chelates are adsorbed on resins of this type and the effect of species as well as for the inert iron, nickel, and ruthenium the coanion have been addressed by Lundgren and Schilt (5). species are also summarized in Table I at various solvent As Kissinger (6) and others (3) have cautioned, it is probably compositions and concentrations of the pairing ion but at a not advisable to view the distribution mechanism in HPLC constant ligand concentration. The VR values given are avas a simple extension of ideas derived from ion-pair partition erages of a number of separate runs and include data where studies or in terms of simple adsorption on the stationary only a single species was injected as well as for mixtures. VR phase. values were reproducible to within the limits of experimental Separation of Labile Chelates. The separation of the errors involved in preparing the solvents and did not appear inert chelates Ru(phen)32+and Fe(phen)l+ appeared to be to change in runs with a number of different metals present about the same when the mobile phase was made M in except for minor shifts due to over lapping peaks when 1,lO-phenanthroline as without the ligand as can be seen in monitored at 265 nm. It was possible to show this effect in Table I. The absorbance of the ligand at this concentration
l T '
-
ANALYTICAL CHEMISTRY, VOL. 54,NO. 2, FEBRUARY 1982 c
B
I
10-3~
z 0
e VI
$
c
0 -
Figure 4. Separation of Zn(phen);+ and Cd(phen)32+: column, p Partisll-SCX; mobile phase, 4:1 CH3CN-H20, M 1,lOphenanthroline, 0.048M LICIO,; detection, 265 nm; temperature 23 OC; (A) 190 ng of Zn, 130 ng of Cd; (B) 110 ng of Zn, 150 ng of Cd.
the cases of the R ~ ( p h e n ) ~and ~ +Fe(phen)32+species by monitoring the elution of these species in the presence of the other chelates at 448 and 512 nm, respectively. Minor shifts in the V, values for these species noted when mixtures were monitored a t 265 nm disappeared when monitored at 448 or 512 nm. This also was a valuable diagnostic tool in confirming peak identity. Flow rates from 0.5 to 8 mL/min were used in obtaining the data in Table I. No significant change in VR values with flow rate was observed but the plate count did decrease somewhat a t flow rates greater than 2 mL/min although the N i ( ~ h e n ) and ~ ~ +F e ( ~ h e n ) species ~ ~ + were still well resolved at flow rates of 8 mL/min for optimal mobile phase compositions. The separation of Zn(phen)32+and Cd(phen)32+at two different zinc to cadmium ratios is shown in Figure 4. The zinc and cadmium were prepared by direct reaction of the Zn2+ and Cd2+ cations with a moderate excess of 1,lOphenanthroline in aqueous solution and injected onto the column. The large peaks near the column volume are observed unless the sample is dissolved in the mobile phase being used but cause no problems if the solute peaks do not elute too near the column volume. The PRP-1 column has not yet been studied as extensively as the p-Partisil-SCX column but the different elution order for the 1,lO-phenanthrolinemetal chelates on this column may
181
prove useful. The Cd(phen)2+species elutes first among the chelates studied followed by the Fe(phen)l+and Ru(phen)?+ species with VR values of 5.87 f 0.1 (n = l l ) , 7.21 f (n = 14) and 8.07, respectively, with a 3:2 acetonitrile-water mobile phase 1.3 X M in 1,lO-phenanthroline and 0.0575 M in LiC104. Zn(phen)t+elutes with V, =I 7.08 mL. The possibility of using this column for the quantitative determination of trace amounts of cadmium and the effect of the ligand concentration in the mobile phase on the retention volumes of the labile complexes are under study. It has been shown that a number of metal chelates of the general type M(phen)t+can be readily separated by IP-HPLC. Columns which have given good separations include the pBondapak-CN (with methanesulfonate but not the perchlorate ion), p-Partisil-SCX and PRP-1. The author was not able to obtain any useful separations using the perchlorate ion as the pairing ion and acetonitrile-water or methanolwater as the mobile phase on p-Partisil-SAX (an anion exchange resin), Zorbax-CN, 1-Bondapak-C18, or the y-Bondapak-CN columns. Little retention and no resolution were observed over a range of mobile phase compositions. Partial resolution of the Fe(phen):+ and Ni(phen)t+species on the Zorbax-CN column was observed with methanesulfonate as the pairing ion but retention volumes were large and the peaks tailed badly. It should be noted that the pressure required to obtain flow rates of up to 2 mL/min on the p-Partisil SCX or the PRP-1 columns with acetonitrile-water as the mobile phase is very small (200-800 psi) and the resolution of these peaks could readily be increased with the use of longer columns. The use of ligand buffered mobile phases for the separation of metal chelates should be possible with other systems but the unique advantages of systems where IP-HPLC can be employed should be noted. The ease with which the retention volumes can be varied by proper choice of the pairing ion concentration with no other change in chromatographic conditions is unique to this method. The development of relatively inexpensive and yet extremely sensitive and selective methods for the trace determination of metals based on the IP-HPLC seems feasible and is currently being studied.
LITERATURE CITED (1) OLaughiin, Jerome W.; Hanson, Russell S . Anal. Chem. 1980, 52, 2263-2268. (2) Eksborg, S.;Schlll, 0. Anal. Chem. 1973, 45, 2092-2100. (3) Tomlinson, E.; Jefferies, T. M.; Riley, C. M. J. Chromefogr. 1978, 759, 315-357. (4) Cassidy, R. M.; Eichuk, S. J . Llq. Chromatogr. 1981, 4 (3), 379-398. (5) Lundgren, J. L.; Schilt, A. A. Anal. Chem. 1977, 49, 974-980. (6) Kissinger, P. T. Anal. Chem. 1977, 4 9 , 883.
RECEIVEDfor review July 24,1981. Accepted October 19,1981.
