Anal. Chem. 1902, 64, 311-315
(36) Lias, S. G.; Bartmess, J. E.; Liebman. J. F.; Holmes, J. L.; Levln, R. D.; ~ a ~ l a r w. d , G. J. phys. chem. Ref. ~ a t awas, suppi pi. 1). (37) Pawliszyn. J.; PhilliDs, J. J . photo&" 1982, 19, 357. (38) Ross, B.; Lacombe', D.; Naikwadl, K. P.; Karasek. F. W. Chemosphere (39)
(40)
ieeo, 20, 1967. Alexandrou, N.; Lawrence, M.; Pawliszyn, J. "Supercritical Fluid Extractkn of Fly Ash Samples for Repld Determinationof Polychbrinated Dibenzo-D-Dioxinsand Dibenzofurans". in Proceedinas of the 1990 htematbnat Symposium on h4easwem6nts of Toxic &d Related POIlutents, Ralelgh, NC, 1990; p 94 U.S. EPA Report NO. EPA1600/9-90/ 026). Alexandrou, N. P. Ciean-up of Complex Environmental Mixtures Using
311
Selecthe Adsorbents and Supercrltlcal Fluids. M.S. Thesis, University of Waterloo, 1991.
R E C for~review July 12,1991. Accepted Odober 24,1991. Financial from the National sciences and Research COunCil of Canada, Imperial oil of Canada, varian and Varian Canada was greatly appreciated. This paper was presented,in during 1991 International symposium on Supercritical Fluid Chromatography in Park city, UT.
Separation of Metal Ions with Sodium Bis(trifluoroethy1)dithiocarbamate Chelation and Supercritical Fluid Chromatography K.E. Laintz, J y a - J y u n Yu,a n d C. M. Wai* Department of Chemistry, University of Idaho, Moscow, Idaho 83843
Bis(trlfluoroethyl)dithbcarbamate(FDDC) forms stable complexes wlth arsenk (As3+) and other metal ions (Bi3+, Co3+, Fe3+,Hg", NP+, Sb3+, and Zn2+) which can be separated by capillary supercrttlcalfluid chromatography (SFC) using CO, as a mobile phase. The fluorinated ligand Is superlor to its hydrogenated form, dlethyldlthlocarbamate (DDC), with respect to thermal staMNty and sdublllty In supercritlcal CO,. Substokhlometrlcsolvent extraction studies showed that the stabllity constants of metal-FDDC complexes were greater than those of their nonffuorlnatedanalogues. Using this FDDC extraction and SFC analysis, separatlon and detectlon of arsenlc specks In the presence of other metals can be achieved with the detectlon limit In the ppb range.
INTRODUCTION A widely used preconcentrationtechnique for trace elements is complexation with the derivatives of dithiocarbamic acid, such as sodium diethyldithiocarbamate (Na(DDC)),followed by a variety of separation and detection methods (1,2).This technique has limited chromatographic use due to the chemical instability and thermal lability of many metal complexes. Since capillary supercritical fluid chromatography (SFC) has been shown to be a method of analysis for thermally labile compounds (3), it is logical to apply this technique to the analysis of metal dithiocarbamates. In prbceeding with this application, we found that many of the complexes formed with metals and Na(DDC) were only partially soluble in supercritical carbon dioxide, as evidenced by poor peak shape due to band broadening, also evidenced by poor reproducibility and chromatographic memory. Neeb and co-workers have shown that substitution of fluorine for hydrogen in DDC, as in the case of sodium bis(trifluoroethy1)dithiocarbamate (NaFDDC), can generally enhance the volatility and thermal stability of the resulting metal chelates (4-8).Substitution of fluorine for hydrogen in some surfactants has been shown to enhance ita solubility in supercritical carbon dioxide (9). Taylor and ceworkers (IO) have investigated the SFC separation of some metal j3-diketonates by using methanol-modifiedC02as a mobile phase. The fluorinated acetylacetone chromium chelate was eluted with a retention time lower than that of ita nonfluorinated 0003-2700/92/0364-0311$03.00/0
analogue. Therefore, these fadors should favor the separation of fluorinated metal chelates in supercritical C02. The behaviors of metal-FDDC complexes in SFC have not been reported in the literature. This paper describes the resulta of our recent study concerning the separation of arsenic and other metal-DDC and -FDDC complexes in SFC using COz as a mobile phase. The application of FDDC extraction followed by SFC as a technique for analyzing metal ions and arsenic species in interstitial water samples is described. A careful survey of the literature, however, shows a lack of information on the stability constants of metal-FDDC complexes. The stability constants of some metal-FDDC complexes determined by this study are also included. EXPERIMENTAL SECTION Instrumentation. A Lee Scientific Model 602 supercritical fluid chromatography system with a Neslab RTE-110 constanttemperaturebath was used for all analyses reported in this work. This system was equipped with a timed-split rotary injection valve and a flame ionization detector (FID). All chromatograms were run using supercritical C02 (Matheson) as the mobile phase and a 5 m 100 pm i.d. by 195 pm 0.d. SB-methyl-100 Superbond capillary column (Lee Scientific). The chromatographic signals were recorded and processed using a HP-3390A integrator. The temperature and density conditions for the analyses were computer controlled and are reported in the Results and Discussion. Reagents. The stock solutions (As,Bi, Co, Fe, Hg, Ni, Sb, and Zn) used in this study were Atomic Spectral Standard Baker Analyzed Reagents from the J. T. Baker Chemical Co. Sodium diethyldithiocarbamate(Na(DDC))was purchased from the Fisher Scientific Co. Other chemicals including chloroform and ethanol were purchased from EM Science. Ammonium acetate buffer was prepared by mixing 120 g of glacial acetic acid (J. T. Baker Ultrapure Reagent) and 134 g of concentrated NHIOH (Aldrich ACS Reagent) and diluting to 1L. The pH value was adjusted by dropwise addition of HN03 and/or NH40H. Deionized water was prepared by passing distilled water through an ion-exchange column (Barnstead ultrapure water purification cartridge) and a 0.2-pm filter assembly (Pall Corp., Ultipor DFA). Sodium bis(trifluoroethy1)dithiocarbamate (Na(FDDC))was syntheaized in our lab according to the procedures outlined in the literature (6). The starting material,bis(Muoroethyl)amine,waa purchased from PCR Research Chemicals. The standard metal-DDC and -FDDC complexes used for calibration were prepared by adding an excess amount of ligand to the metal solutions at the appropriate pH (7). The resulting precipitates were extracted into chloroform, and the organic phase was washed with deionized 0 1992 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 3, FEBRUARY 1, 1992
water after phase separation. Purification of the metal complexes was done using recrystallization from a chloroform/ethanol solution (1:lv/v). Other chemicalsused in the synthesis, including sodium amide, carbon disulfide, and potassium hydroxide, were all obtained from Aldrich Chemical Co. All containers used in the experiments were acid-washed, rinsed several times with deionized water, and dried in a Class 100 clean hood. Interstitial waters were obtained by pressure squeezing sediments with an Amicon Model 402 gas squeezing apparatus. The sediments were collected from the Coeur d‘Alene River near Smelterville, Idaho. The water was then filtered through a 0.45-pm Millipore membrane filter and stored at 4 OC overnight in a 1-L polyethylene bottle prior to use. Extraction Procedures. Prior to dithiocarbamateextraction, the filtered interstitial water samples were first shaken with chloroform to remove organicmaterials. To extract metal species, one aliquot of a sample was adjusted to pH 3 using an acetate buffer. About 200 mg of Na(FDDC) diasolved in 2 mL of deionized water was usually added to 20 mL of a sample solution. Chloroform (2 mL) was then added to the solution and the mixture was shaken vigorously for 20 min. After phase separation, the organic phase was transferred into a 1-drampolyethylenevial and was allowed to evaporate to dryness. Exactly 100 pL of dichloromethane was then added to the vial to redissolve the extracted metal chelates for SFC analysis. According to this extraction procedure, a preconcentration factor of 200 can be achieved. Pentavalent As5+and Sb5+in water samples cannot be extracted by Na(FDDC) according to the procedure given above. Prior to extraction, the reduction of Asw and Sb5+to their trivalent states was conducted by adding 1 mL of 0.2% K1 and 0.5% Na&03 solution at pH 2. Quantification of the pentavalent species is then accomplished by subtraction of trivalent concentrations from the total concentrations. Procedures for Determination of the Stability Constant of Metal-FDDC Complexes. The stability constant of As(FDDC)3was obtained by extracting a substoichiometric amount of As3+with a mixture of equal amounts of Na(DDC) and Na(FDDC). From the relative concentrations of As(DDC)~and As(FDDC)3observed from the competition experiment, the stability constant of As(FDDC)~was calculated on the basis of the value of AS(DDC)~The stability constants of other metal-FDDC complexes were determined by comparison with that of As(FDDC)3using the substoichiometricextraction technique. In general, 5 mL of pH 4.2 buffered aqueous solution of known concentrations M each) was mixed with 5 mL of of Mn+and As3+(1.0 X freshly prepared FDDC solution in different concentrations(vary to 3.5 X loF3M). The solution was shaken for from 1.0 X 10 min. A 10-mLaliquot of chloroform was added to the solution, and the mixed solution was shaken for another 10 min. After waiting for another 5 min to allow phase separation, 5 mL of the aqueous phase was pipetted into a plastic vial for atomic absorption or neutron activation analysis (NAA). Sample Irradiation and Counting in NAA. The details of sample-sealing procedures for neutron irradiation are given elsewhere (11). Standards and samples, made of 1-mLsolutions containing proper concentrations of metals, were heat-sealed in the same 2/5-drampolyethylene vials. All standards and samples were irradiated for 2 h in a 1-MW TRIGA reactor at a steady neutron flux of 6 X 10l2n/(cm2 s) followed by a 12-h cooling period. Each sample was counted using a large-volumecoaxial ORTEC Ge(Li) detector with a resolution of about 2.3 keV at the 1332-keVy ray from B°Co. RESULTS AND DISCUSSION SFC Separation of Metal-FDDC Complexes. SFC using alcohol-modified COzas the mobile phase for elution of some metal-DDC complexes has been studied using packed columns with marginal success (12,13). However, the use of capillary SFC with pure COzas the mobile phase to separate and detect metal-FDDC complexes has not been reported in the literature. The chromatographic Conditionsused in our experiments are as follows: an oven temperature of 100 OC with an initial COzpressure of 100 atm, followed by a 6.5-min hold time with a pressure ramp of 4.0 atm/min to a final pressure of 200 atm. The sample injection time was 0.1 s, which amounts to a
c22
5
10
15
20
25
30
Time ( m i n )
Flgure 1. Chromatogram produced using a Lee Scientific 5 m X 100 pm i.d. SBmethyCl00 superbond capillary column. SFC conditions: 80-nL sample injected at 100 OC oven temperature with a hold time of 6.5 mln at 100 atm followed by a 4.0 atm/min ramp to 200 atm. h
calibrated 80-nL sample injection. The FID temperature was 325 “C. Under these conditions, separation and detection of some metal dithiocarbamate complexes such as As(DDC),, Ni@DC)z, Pb@DC),, and Z~I@DC)~ were poasible. However, judging from the poor reproducibility of results and broad peak shapes,these particular metal complexes apparently have a low solubility in supercritical carbon dioxide. These difficulties were further compounded by sample decomposition and retention within the column, resulting in chromatographic memory and subsequent column contamination. Quantitative analysis of metal-DDC complexes by SFC is not practically feasible according to our experiments. Fluorination of the ligand has drastically improved the chromatogaphic behavior of these metal chelates. Figure 1 illustrates this point with a comparison of a sample analyzed by capillary SFC containing the same concentration (6 X lo4 M) of As(FDDC)~and AS(DDC)~,with docosane (C,H&) being used in this case as an internal standard. The AS(DDC)~peak is typical of metal-DDC complexes, typically having a somewhat poor chromatographic performance. On the other hand, the As(FDDQ3 peak is sharp and well-defined, with a much shorter retention time relative to the corresponding DDC complex. Part of this improvement is assumed to be due to enhanced mobilephase interactions. Furthermore, the chromatographic results of As(FDDC)~were reproducible without any of the column contamination problems that were encountered using DDC complexes.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 3, FEBRUARY 1, 1992
313
d
-
5
I 0
I
1
1
5
10
15
I
I
70
25
1
Time (min)
ngve 3. Chromatogam of a FDDC solvent extracted interstltlal water sample using the same conditions as Figure 1: (a) Na(FDDC); (b) zn(FDDC),; (c)WFDDC),; (d) WFWCk: (e) As(FDDCh; (0 Sb(=k; (9) Mn(FDW,.
Table I. Comparison of Analytical Results for an Interstitial Water Sample Obtained by SFC, NAA, and ICP-AES Methods metal species As3+
As5+ Astotal
Sb3+ Sb5+ Sbtotal cu2+ Fe3+
Zn2+
concentration, pg/Lu SFC NAA ICP-AES 45 f 6 25 f 7 70 f 5 27 f 6 17 f 8 44 f 6 47 f 4 165 f 14 188 k 7
40 f 6 25 f 7 65 f 4 25 f 1 20 f 4 45 f 4
50 f 1 158 f 4 182 f 5
aAs5+and Sb5+were determined by the quantity [Mtatal- M3+].
Figure 2 shows a series of metal-FDDC chelates that were separated and detected, which includes Zn-, Ni-, Co-, Fe-, Hg-, As-, Sb-, and Bi(FDDC) complexes. This shows the ability to separate and detect arsenic from a mixture of metal complexes. The detection limit of these metal complexes is generally on the order of 1 ppm of chelate dissolved in the organic phase, based on an 80-nL injection volume. The extraction procedure serves as a preconcentrationstep for SFC analysis. With a preconcentration factor of 100-1000, this technique is suitable for analyzing elements in natural waters. This method was subsequently employed for the determination of arsenic, antimony, and other metals in an interstitial water sample obtained from river sediment. The water sample (20 mL) was extracted following the procedures described in the Experimental Section. The SFC chromatogram of the extracted As3+ion is shown along with extracted Zn2+,Cu2+,Fe3+,Sb3+,and Mn2+in Figure 3. Manganese(I1) was the only metal not quantified due to the inability to achieve quantitative extraction. The water was found to contain a significant amount of As and Sb in addition to Zn, Cu, and Fe. The ratio of As3+/As5+in the interstitial water is high, suggesting that the pore water in the sediment was in a rather reduced environment. The overall results summarized in Table I agree with those determined by solvent extraction followed by NAA and ICP-AES.
15
10
Time ( m i n ) Flgure 4. Chromatogram produced using a L e e Scientific 5 m X 100 pm 1.d. SBmethyClOO superbond capillary column: (a) Na(FDDC);(b) DMA(FDDC); (c) MMA(FDDC),; (d) As(FDDC),. Condltlons: 80-nL sample injected at 100 O C oven temperature wnh a hold tlme of 6.5 min at 100 atm followed by a 15.0 atm/min ramp to 200 atm.
Of further interest is the fact that arsenic can exist in aqueous environments as inorganic species, arsenite and arsenate, and as organic species, including CH3AsO(OH), (MMA), (CH3)2AsO(OH)(DMA), etc. (14). Determination of these arsenic species is important because the toxicity, bioaccumulation, and migration of arsenic in the environment depend on its chemical forms. The extraction of MMA and DMA requires reduction with a mixture of potassium iodide, sodium thiosulfite, and sulfuric acid, which converts the organoarsenicals to CH3As12and (CH3)2AsI,respectively. The iodides can be extracted with FDDC into chloroform as CH3As(FDDC),and (CH,),As(FDDC). The resulting FDDC chelates, As(FDDC),, CH3As(FDDC),,and (CH,),As(FDDC), can be separated by SFC, as shown in Figure 4. The recovery is usually >go%, and the detection limit of these arsenicFDDC complexes by FID under our SFC conditions is about 1ppm on the basis of a signal to noise ratio of 3. Incomplete recovery of the organoarsenicals is attributed to incomplete reduction of those species present. Stability Constant of Metal-FDDC Complexes. The determination of the stability constant of As(FDDC), was done in a competition experiment where As3+ was added in a substoichiometric amount to a mixture of equal amounts of Na(DDC) and Na(FDDC). In this experiment, the concentration of each ligand was 2.1 X lo-, M, while the concentration of As3+was 2.1 X low3M. After extraction, the organic phase was analyzed by SFC to determine the relative amounts of As(FDDC), and As(DDClB. The relative stability constants of the two arsenic chelatea can be calculated from the following equilibrium relations: As3+ + 3FDDCAs3+
+ 3DDC-
-
As(FDDC)~
(1)
As(DDC)3
(2)
K,l/K,, =
([As(FDDC)~I/[A~(DDC)~IH[DDC-I /[FDDC-Il3 (3) where K,, and K,, are the stability constants of As(FDDC)~ and AS(DDC)~, respectively. In this case, the equilibriumratio {[As(FDDC),]/[As(DDC),]J was determined to be 2.96, and the { [DDC-I/ [FDDC-]$ equilibrium ratio was determined to be 1.71 by subtraction of the molar concentrations of complexed ligand from the starting ligand concentrations. Recently, K,, has been determined in our laboratory as 7.1 X loz3. From the relative concentrations of the two arsenic complexes, K,, can be calculated from eq 3. The value of KB1 determined from this experiment is 3.6 X 10". The stability constants for some other metal-FDDC complexes were also
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 3, FEBRUARY 1, 1992
Table 111. Solubility of Selected Metal-FDDC and Metal-DDC Chelates in Supercritical C02 at SO OC and 100 atm
Table 11. Stability Constants of Some Metal-FDDC Complexes Determined by Substoichiometric Extraction M(FDDC), 1/n log K B
metal
K,
As3+
3.60 X loz4 2.67 X lOl9 1.65 X loZ5 7.63 X 1015
Pb2+ Sb3+ Zn2+
8.19 f 0.04 9.71 f 0.01 8.74 f 0.02 7.94 f 0.01
M(DDC), l l n log KB
metal chelate
7.70 f 0.02 9.51 f 0.79" 8.50 f 0.02 7.42 f 0.87"
Ni(DDQ Ni(FDDC& Co(DDC)3 Co(FDDC)s
'Average of KRvalues obtained from refs 15-20.
+ 3FDDCMn+ + nFDDC-
--
As(FDDC)~
(1)
M(FDDC),
(4)
KBM = D,/[FDDC-]"
KBln/K,9M3 = DAsn/DM3
(6) (7)
Where n is the charge of the cation, D, and D M are the distribution coefficients defined as follows: D A ~ [As(FDDC),I,rg/[As3+laq
(8.5 f 1.0) x (7.2 f 1.0) X (2.4 f 0.4) X (8.0 f 0.6) X
10-7
lo4 lo4
lo4
rinated metal chelates in supercritical COz are currently under
determined by the substoichiometric extraction technique. For this experiment a substoichiometricamount of FDDC was added to a solution containing equal concentrations of As3+ and another metal ion, Pb2+, Sb3+,or Zn2+. The relative stability constants for the metal chelates can be calculated from the following equilibrium relations: As3+
solubility, mol/L
(8) (9)
Using the chromatographically determined value for KBlof 3.6 X IOz4,KBM for the other metals can be calculated from eq 7. These values are usually expressed as (l/n) log KBMand are summarized in Table I1 along with the values for the metal-DDC Complexes for comparison. The reported values for the metal-FDDC complexes are based on triplicate analysis. The values listed in the table for Pb(DD(& and Z I ~ D D Cwere ) ~ averaged from reported literature values from 1964 to 1980 (15-20), with standard deviations based on those averages. The values for As(DDC)~and Sb(DDC)3were recently determined in our laboratory. The results show that the stability of metal complexes is generally enhanced by substitution of fluorine in the DDC ligand. Solubility of Metal-FDDC Complexes in Supercritical COz. The solubilities of metal chelates can be measured spectroscopically, as described by Laintz et al. (21). Unfortunately, some metal-FDDC complexes such as As(FDDC)3 do not have distinct absorption peaks in the UV-vis region which can be used for quantitative determination. However, some DDC complexes with transition metals are known to have strong charge-transfer absorptions in the visible region (22). For example, Co(DDC), and Ni(DDCl2 show strong absorption peaks in supercritical COz. Substitution of F for H in the ligand shifts the charge-transfer absorption peaks to lower energy. For example, the chargetransfer absorptions of Ni(DDC)z at 432 and 381 nm are shifted to 468 and 404 nm for Ni(FDDC)z. These absorption peaks were used to measure the solubilities of the DDC and FDDC complexes in supercritical COz. At 50 OC and 100 atm, the solubilities of the Co and Ni complexes in supercritical C02obtained from our experiments are summarized in Table 111. The enhancement in solubility is about 2-3 orders of magnitude for the fluorinated dithiocarbamate complexes. The significant increase in solubility is an important factor favoring the separation of fluorinated metal dithiocarbamate over the nonfluorinated ones in SFC. The solubilities of other fluo-
investigation.
CONCLUSION This study has demonstrated that bis(trifluoroethy1)dithiocarbamate is an effective chelating reagent for the extraction of arsenic and other metal species in aqueous solutions. The resulting FDDC chelates are more stable and soluble in supercritical carbon dioxide relative to the nonfluorinated DDC chelates. Simultaneous determination of these metal species can be achieved with a capillary column SFC system. Neeb and co-workers have reported the separation of a number of metal-FDDC complexes described in this study (except arsenic species) by gas chromatography (4, 5, 7). The separation of As(FDDC)~from other metal complexes by GC has been described in a recent report by Yu et al. (23). A discussion of the thermal stability of metal dithiocarbamate complexes has also been given by these authors. SFC, however, has the advantage of much lower injection temperature (room temperature) and oven temperature (100 "C) which reduces the risk of sample decomposition. HPLC has also been used to separate some metal-DDC complexes, but the separation is very sensitive to solvent composition and the results are often not reproducible (24-26). Our attempt to separate FDDC complexes by both normal-phase and reversed-phase HPLC showed poor resolutions and irreproducible results for several metals. The difficulty is suspected to be partially caused by solubility and instability problems of metal chelates in the mobile phases. Metal-DDC complexes are sparsely soluble in water and decompose depending on the pH of solution. These problems are not encountered in SFC using COPas a mobile phase. In the case of separating thermally and chemically labile metal chelates, this fluorinated chelating agent in combination with SFC separation appears to provide a rapid and sensitive method of analyzing areenic species and other metals in aqueous solution. ACKNOWLEDGMENT This material is based upon work supported by the Idaho EPSCoR Program of the National Science Foundation under Grant No. RII-8902065. REFERENCES (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17)
Huianlckl H. Talenta 1987, 1 4 , 1371. Lo, J. M.; Yu, J. C.; Hutchlson, F. I.; Wal. C. M. Anal. Chem. 1982, 54, 2536. White, C. M.; Houck, R. K. HRC & Gc,J . H@h Resolid. Chromatogr. Chmnatogr. Commun. 1985, 8 , 293. Neeb. R. Pure Appl. Chem. 1982, 54, 847. Schaller, H.; Neeb, R. Fresenius' Z . Anal. Chem. 1988, 323, 473. Tavlerdls, A.; Neeb, R. Fresenlus' Z . Anal. Chem. 1978, 292, 135. Schneider. H.; Neeb, R. Fresenius' Z . Anal. Chem. 1978, 293, 11. Schaller, H.; Neeb. R. Fresenius' 2.Anal. Chem. 1987, 327, 170. Consanl, K.; Smith, R. J . Supercrf. Nu& 1990, 3, 51. Ashraf-Khorassanl. M.; Hellgeth, J. W.; Taylor, L. T. Anal. Chem. 1987, 59.2077. W, W. M.; Shah, N. K.; Wai. C. M. Anal. Chem. 1988, 58, 110. Blckmann, F.; Wenclawiek, B. Fresenlus' Z . Anal. Cbem. 1984, 379, 305. Blckmann, F.; Wenclawlak. B. Frensenlus' Z . Anal. Chem. 1985, 320, 261. Andreae, M. 0. Anal. Chem. 1977. 49, 820. Still, E. F h . K e m i s t s a m l W . 1984, 73, 90. Stary, J.; Kratzer, K. Anal. CMm. Acta 1968. 4 0 , 93. Scharge, R. R.; Sastri, V. S.; Chakrabartl, C. L. Anal. Chem. 1979, 4 5 , 413.
Anal. Chem. 1992, 64, 315-319 (18)
Ooms,P. C. A.; Brinkman. U. A.; Das, H. A. R a d l o d " . Radiaenel. Lett. 1977, 31, 317.
(19) BaJo,S.; Wytlenbach, A. Anal. Chem. 1070, 57. 376. (20) Shen, L. H.; Yeh, S. J.; Lo, J. M. Anal. Chem. 1980, 52, 1882. (21) Laintz, K. E.; Wai. C. M.; Yonker, C. R.; Smith, R. D. J . Supercrft. Flu& 1991, 4 (3). 194. (22) Bode, H. FreseniuP' Z . Anal. Chem. 1955, 144, 165. (23) Yu, J. J.; Wai. C. M. Anal. Chem. 1991. 63, 842.
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(24) Schwedt, 0. Chrometograph& 1979, 12. 290. (25) Bond, A. M.; Wallace, G. G. Anal. Chem. 1989. 55, 718. (26) Ichinoki, S.; Yamazaki, M. Anal. Chem. 1985, 57, 2219.
RECEIVED for review August 1,1991. Accepted October 31, 1991.
Formation in an Aqueous Matrix and Properties and Chromatographic Behavior of I-Pyrenyldiazomethane Derivatives of Methylmalonic Acid and Other Short-Chain Dicarboxylic Acids J8rn Schneede* and Per Magne Ueland The Department of Pharmacology and Toxicology, University of Bergen, Armauer Hamens hus, N-5021 Bergen, Norway
MethytMknic sdd (MMA) and 801118 shortdaln dlcarboxyk acids were derivatized with the fluorescent iabellng reagent l-pyrenyldlazomethane (PDAM) in an aqueous matrix. The derivatization of MMA and ethyimaionic acid (EMA) proceeded and leveled otf rapklly at b w pH, whefeas at akallne pH, the fluorescent derivative was progrdvely f m e d over ylekl was a perkd of more than 24 h, and ~ f i u o r e s c e n c e obtained. The PDAM esters of MMA and EMA had an excitatbn maximum at 340 nm and emlsdon m a m a at 376 and 395 MI. They wem stable for days h aqueous media at room temperature. The MMA derivative was Identified as l-pyrenybnethyl methyhralonatemonoester by mass spectrometry, and formation of l-pyrenyimethyl methylmaionate diester could not be demonstrated. The free carboxylk acid moiety and the ionization of this group may explain the unlque chromatographic properties of the MMA derivative, 1.e. a marked increase in the capacity factor In a reversed-phase system by decreashg the pH of the moblle phase. The MMA derivatlve was separated from PDAM esters of several other short-chain dicarboxylic acids by a acetonitrile gradlent in formate buffer, pH 2.5. These data may form the basls for the construction of automated MMA assays involving derive tkatkn In aqueous media followed by liquid chromatography.
INTRODUCTION Several chromatographic techniques have been developed for the determination of carboxylic acids because of the importance of these compounds in normal metabolism and cellular function, as well as their role in several diseases. Among the carboxylic acids, great interest has recently been focused on the short-chain dicarboxylicacid methylmalonic acid (MMA). Its concentration in extracellular media like plasma and urine may serve as an indicator of intracellular cobalamin function (1). Carboxylic acids including MMA have been determined by GC or GC/MS (2-4). These methods often require extensive sample purification and derivatization prior to chromatography ( 3 , 4 ) . Recently, several liquid chromatographic procedures have been described, which involve precolumn derivatization with either a chromophor (5,6)or fluorophor (7). 0003-2700/92/0364-0315$03.00/0
Fluorescence derivatization often creates a high sensitivity of the assay, and several fluorotags reacting with carboxylic groups have been developed. These include arylbromo- and arylchloromethanes, acylbromomethanes, fluorescent alcohol or amines, and aryldiazomethanes (7). These reactions are often performed in aprotic solvents because of the low reactivity of the carboxylic moiety in water. Therefore, tedious extraction procedures are often necessary, but this may be avoided by using phase-transfer techniques or more recently by micellar catalysis (8). 1-Pyrenyldiazomethane (PDAM) is a newly synthesized aryldiazomethane which offers several advantages as a fluorescent labeling reagent of carboxylic acids for liquid chromatography. Both PDAM and the reaction product are stable. PDAM readily reacts with monocarboxylic acids at room temperature without a catalyst. The reaction can take place in both protic and aprotic solvents, and the products are intensely fluorescent esters (9). We studied the reaction of PDAM with MMA and some other saturated short-chain dicarboxylic acids (C < 5 ) in aqueous media. Under these conditions, only one carboxylic group of these acids reads with PDAM, and the other remains underivatized. Stable fluorescent products are formed, and the free carboxyl group results in unique chromatographic properties of the monoesters. The retention of these compounds on reversed-phase and anion-exchange columns is influenced by the ionization of the free carboxyl group and thereby the pH of the mobile phase.
EXPERIMENTAL SECTION Chemicals. PDAM was purchased from Molecular Probes, Inc. (Eugene, OR). It (2.5 mg/mL) was dissolved in ethyl acetate and stored at -20 "C. This solution was freshly prepared each 14 days. PDAM must be regarded as potentially hazardous, and skin and eye contact should be avoided. Mechanical ventilation and respiratory protection are recommended. MMA, ethylmalonic acid (EMA),malonic acid, fumaric acid, a-ketoglutaticacid, and dimethylmalonicacid were obtained from Aldrich Chemical Co. (Milwaukee, WI), and succinic acid was obtained from Sigma Chemical Co. (St. Louis, MO). Methanol (HPLC grade), acetonitrile (HPLC grade), and ethyl acetate were from Merck (Darmstadt, FRG).We wed double-distilledwater which was further purified on a Milli-Q-plus ultra-pure water system (Millipore Corp., Bedford, MA). Packing material for reversed-phaseliquid chromatography,Hypersil(3 pm), was from 0 1992 American Chemical Society
