Anal. Chem. l W 5 , 57,279-283 (3) Iler, R. K. “The Chemistry of Silica”; Wiley: New York, 1979. (4) Iler, R. K. J . Chromatogr. 1961, 209, 341. (5) Rollhgs, J. E.; Base, A.; Caruthers, J. M.; Okos. M. R.; Tsao, G. T. f d y m . frepr.. Am. Chem. Soc., Dlv. folym. Chem. 1981, 22. 294. (6) Rochas, C.; Domard, A,; Rinaudo, M. Eur. folym. J. 1980, 16, 135. (7) BarthiH. G.; Regnler, F. E. J. Chromatogr. 1980, 192, 275. (8) Cooper, A. R.; Matzinger, D. P. J. Appl. folym. Sci. 1879, 23, 419. (9) Flory, P. J. “Principles of Polymer Chemistry”; Corneil Unlversity Press: Ithaca, NY, 1953; pp 605-61 1. (10) Casassa, E. F.; Tagami, Y. Macromoiecules 1989. 2, 14. (11) Grubisic, 2.; Rempp, P.; Benoit, H. J. fo/ym. Sci., Part B 1967, 5 , 753. (12) Dubin. P. L.: Wrlaht. K. L.; Koontz, S. L. J. fo/ym. Sci. Chem. Ed. 1978, 15, 2047.(13) Haller, W. Nature (London) 1985, 206, 693. (14) Hailer, W.; Basedow, A. M.; Konig, B. J. Chromatogr. 1977, 132, 367. (15) Klein, J.; Westerkamp, A. J. folym. Sci. Chem. Ed. 1981, 19, 707. .
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(16) Klm, C. J.; Hamlelec, A. E.; Benedek, A. J . Liq. Chromatogr. 1982, 5 , 425. (17) Basedow, A. M.; Ebert, K. H.; Ederer, H. J.; Fosshag, E. J. Chromatogr. 1980, 792, 259. (18) FUOSS,R. M.; Strauss, U. P. J . folym. Sci. 1948, 3 , 602. (19) Casassa, E. F. J. fhys. Chem. 1971, 7 5 , 3929. (20) See, for example: Moore, W. J. “Physical Chemistry”; Prentlce-Hall: Englewood Cliffs, NJ, 1972; pp 510-513. (21) Hiemenz, P. C. “Principles of Colloid and Surface Chemistry”; Marcel Dekker: New York, 1977; p 373. (22) Loeb, A,; Overbeek, J.; Welrsma, P. “The Electrical Double Layer Around a Spherical Coliiod Particle”; M.I.T. Press: Cambridge, MA, 1961. (23) Bolt, G. H. J. fhys. Chem. 1957, 6 1 , 1166.
RECEIVED for review July 6, 1984. Accepted September 10, 1984.
High-pressure, Aqueous, Size-Exclusion Chromatography of Copper(I I) Complexes of Poly(aminocarboxy1ic acids), Catechol, and Fulvic Acids Using Reverse-Pulse Amperometric Detection Mary L. Adamid and Duane E. Bartak*
Department of Chemistry, University of North Dakota, Grand Forks, North Dakota 58202
High-performance, size-exclusion chromatography (SEC) on copper(II)complexes of a series of organic ligands lncludlng (1) poly(amlnocarboxy1lc aclds) (NTA, EDTA, and DTPA), (2) ctlrlc acid, (3) catechol, and (4) waterderlved fulvlc acid was Investigated. Two commerclally available SEC columns, a TSK-2000 SW and a Synchropak GPC-100, were tested In a modified Hummel-Dryer mode In which the eluent contains copper( 11) to prevent dlssoclatlon of the labile metal complexes. An electrochemical flow cell was used as a detector to ldentlfy and quantify the copper(I1) complexes. The electrochemical current measurements were made by uslng a reversepulse mode to minimize oxygen Interferences at the detector. A detectlon llmlt of 40 ng was measured for the CU-EDTA complex. Copper-fulvlc acid complexes were tested, and a detection llmlt of 1.2 pg was obtalried. The above techniques have the potentlal appllcablllty In the determlnatlon of the complexatlon capacity of natural water samples.
Recent developments in column packings for high-performance size-exclusion chromatography (SEC) have led to their use on aqueous systems. In particular, the development of controlled pore silica beads, on which hydrophilic groups have been covalently bonded, has resulted in several aqueous size-exclusion columns, which are commercially available. Some of these include (1)TSK Type SW (Toyo Soda), which has covalently bound hydrophilic groups containing OH groups, (2) Synchropak (Synchrom), which contains glycerylpropylsilyl groups covalentlybonded to silica supports, (3) Present address: Westinghouse I d a h o Nuclear Co.,
4000, I d a h o Falls, ID 83403.
P.O.Box
pBondage1 (Waters Assoc.), which contains polyether moieties bonded to silica, (4) LiChrosorb Diol (Merck), which like Synchropak contains glycerylpropylsilyl groups covalently bonded to silica, and (5) Vydac (Separatives Group), which contains octadecyl groups (i.e., typical CI8 reverse-phase column) that are end-capped in a manner (proprietary) such as to minimize adsorption on the silanol (SiOH) group of silica. The above columns have been applied to several water-soluble biological systems including proteins, enzymes, nucleic acids, and polysaccharides (1,2). In addition, these columns have been recently tested with humic substances including humic and fulvic acid ( 3 , 4 ) . Size-exclusion chromatography of humic substances had been previously carried out on soft gels such as Sephadex (5-7). However, the soft gels are operated at lower pressures with the result that the separation is timeconsuming. Furthermore, particle sizes of the soft gels are larger and less uniform, resulting in reduced column efficiency. We have previously reported on the use of Sephadex gels to separate and quantify metal complexes using a modified Hummel-Dreyer technique, which utilizes an eluent containing excess metal ions to prevent dissociation of labile complexes’during the separation process (8). Poly(aminocarboxylic acids), citric acid, and fulvic acids were studied using copper(I1) in the eluent. However the technique had limitations due to (1)long analysis times, (2) relatively low column efficiency (typical HETP = 1mm), and (3) probable adsorption of copper-fulvic acid complexes, which contain hydroxy and carboxy groups, on the dextran-based Sephadex packing. We report herein on the use of two commercial, high-performance SEC columns for the separation of metal complexes in the modified Hummel-Dreyer mode. An improvement in analysis time and column efficiency will be demonstrated. Furthermore, the adsorption of a model natural water ligand, catechol, is significantly diminished on these HPLC columns. A detection limit for water-derived
0003-2700/85/0357-0279$01.50/00 1984 American Chemlcal Society
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fulvic acid is determined, and the quantification of fulvic acids in a natural water sample is demonstrated. The metal detector for the chromatographic system consists of an electrochemical flow cell operated in the reverse-pulse amperometric mode.
EXPERIMENTAL SECTION Instrumentation. The chromatographicsystem consisted of a Milton Roy minipump equipped with pulse dampeners (Waters Assoc.) and a Rheodyne Model 7125 syringe-loadingsample injector with 50- and 500-pL sample injection loops. Two highperformance aqueous size-exclusion columns were tested: (1) Spherogel TSK-2000 SW column, 10-pm particle size (0.75 cm X 30 cm) (Beckman Instruments) and (2) SynChropak GPC 100 column, 10-pm particle size (0.46 cm X 25 cm) (Alltech Assoc.). The metal detector was a flow-through electrochemical cell (Princeton Applied Research (PAR) Model 310), which was used in a dropping mercury electrode (DME) mode. Potential control of the electrochemical cell and amperometric measurements at the DME were made with a PAR Model 174A polarographic analyzer. Reverse-pulse amperometry (rpa) was carried out by operating the PAR 174A in a pulse mode (9). The potential program for the rpa experiment using copper as the metal to be detected was set up by (1)applying an initial potential of -0.75 V, (2) scanning in the positive direction to give a final potential of 0.0 V, and (3) then setting the PAR 174A switch to HOLD. The result is a potential pulse with an amplitude of 0.75 V (-0.75-0.00 V) and pulse width of 57 ms, which is applied to each successive mercury drop during the last 57 ms of the lifetime of the drop. The PAR 174A in the pulse mode measures the current during the last 17 ms of each pulse (as in normal pulse polarography). The measured current was recorded on a Houston Omniscribe strip chart recorder to produce the chromatograms. Electrodes. The DME drop area in the medium-size setting cm2. The glass capillary on the PAR 310 was found to be 1.7 X for the DME was siliconized at least monthly with a 5% solution of dichlorodimethylsilane in CC4. The reference electrode was a Ag/AgCl electrode with saturated KC1 and AgCl as the filling solution, which was isolated from the contents of the cell with a porous glass salt bridge. The porous glass separator in the reference electrode bridge was changed monthly. A platinum wire was used as the counter electrode. Chemicals. All chemicalswere analytical reagent grade. The mercury used in the DME was triply distilled. All water used was distilled and subsequently passed through a Millipore Q purification system. The eluent buffer used in most experiments contained 0.05 M tris(hydroxymethy1)aminomethane acetate adjusted to pH 7.4 with acetic acid, 0.10 M NaN03, and 1.6 X loi M (10 ppm) copper. The water-derived fulvic acid (WFA) was isolated and purified by using a procedure previously reported by Weber (10). Water, which was light brown (weak tea) in color (pH 7.8, less than 10 ppm BOD), from the English Coulee (a tributary to the Red River) in Grand Forks. ND. was DumDed through two (45 cm X 4.5 cm) columns packed with Amberlke IRA-4k anion-exchange resin in the hydroxide form. The water was propelled through the two columns by a peristaltic pump (Bio-Rad)at a rate of 0.1 gal min-’ for 5 days. The isolated, resultant humic and fulvic acids produced a dark brown coloration on the column packing. The humic and fulvic acids were then eluted from the columns by passing 2 M NaCl through columns until no additional coloration was noted in the eluent. The NaCl solution of humic and fulvic acids was acidified to pH 1,whereupon the humic acid precipitated out and was separated. The solution containing fulvic acid (WFA) was then passed through a column packed with hberlite XAD-2 resin (a nonionic polymeric adsorbent) at a flow rate of 3 mL min-’ to adsorb the WFA. The WFA was then desorbed from the column by using a 1% NaOH solution,which effectively forms the sodium salt of fulvic acid. The resultant eluent was then recycled through the column until a final eluent with a pH 8 was obtained. The eluent containing the sodium salt of fulvic acid was then passed through a Rexyn 101(H) cation-exchangeresin (hydrogen form) to remove the sodium. The eluent from the cation-exchange column was subsequently filtered through a 0.45-pm Millipore filter and evaporated to 250 mL on a rotatory evaporator. The remaining solutionwas freeze-dried to a brown powder with a yield of approximately 3 g.
RESULTS AND DISCUSSIONS TSK-2000 S W GPC Column. The TSK-2000 SW is a high-performance aqueous SEC column, which has packing that is silica-based and contains hydroxyl groups; however, the composition is proprietary. The molecular weight range for this column has been shown to be 500-15000 daltons for polyethylene glycols and 1000-30000 daltons for dextrans (12). This molecular weight range would appear to be reasonably consistent with possible metal complexes which could be present in natural water systems. Saito and Hayano have used a TSK 3000 SW column, which had a reported range of 1000-35000 daltons for polyethylene glycols ( I I ) , in a study on humic materials from marine sediment (3). Their data indicated that separation of humic acid from fulvic acid was possible if the eluent contained 0.1 M NaCl so as to decrease coulombic replusion between the gel and solute. In addition, their data showed that the retardation of solutes due to adsorption was not important on the TSK column, which was in contrast to the earlier humic acid work on Sephadex gels (5). However, other data on a TSK-2000 SW column indicated that although basic amino acids were not adsorbed on the gel, some aromatic amino acids and nucleic bases were strongly adsorbed on the gel (12). Although the exact structure of the TSK-SW gels has not been published, it has been reported that the support is a rigid silica gel with surface-containing hydroxyl groups (13). These “hydroxyl groups” could be the cause of the observed adsorption effects. A series of poly(aminocarboxy1ic acids) were tested on the TSK column to determine the performance of these columns when used in a modified Hummel-Dreyer mode. The total metal concentration of the eluent was monitored by an electrochemical flow cell in the reverse-pulse amperometric mode. Reverse-pulse amperometry was utilized to minimize oxygen interference and to maximize sensitivity of the detector (9). The resultant chromatogram ideally consisted of a peak that is the result of the metal complex and a trough, which results from the depletion of metal ions in the formation of the complex (14,15). Figure 1A is a modified gel permeation chromatogram for EDTA on the TSK column using 10 ppm copper(I1) in the eluent. In order to reduce coulombic replusion (Donnan effect) between the packing and charged solutes, 0.1 M NaN03 was added to the eluent. The chromatogram shows the ratio of the elution volume to void volume (V,/ V,) to be 1.10 for the Cu-EDTA complex. The retention volume of the trough is 17.7 mL, which is approximately equal to the total volume (V, = 17.8 mL) of the column. The area of the trough, which represents the depleted copper(I1) from the eluent, is 98% of the peak area for the complex peak. The resolution for a mixture of poly(aminocarboxy1icacids) (DTPA (diethylenetriaminepentaacetic acid), EDTA, and NTA (nitrilotriacetic acid)) on the TSK column is illustrated in Figure 1B. The resolution of metal complex peaks for this mixture of copper(I1) complexes is approximately the same on TSK as on the less efficient Sephadex G-25 column. However, the trough or metal depletion band is better resolved from the copper(I1) complex peaks on the TSK column. The optimum parameters for the quantitation of the poly(aminocarboxylic acids) were determined using EDTA as a model. The dependence of detector response for the Cu-EDTA complex showed a gradual decrease in peak area with increasing flow rates from 0.3 to 1.1 mL/min; a t flow rates greater than 1.0 mL/min the peak area remained constant within experimental error. This behavior for the response of the electrochemical detector is consistent with variations in mass transport in an electrochemical cell. Convection and diffusion are predominant at the slower flow rates while convection is the sole mode of transport at the more rapid flow rates in the electrochemical cell, The efficiency of the TSK
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Figure 1. (A) Modified size-exclusion chromatogram for 0.12 bmol of EDTA on a TSK-2000SW column. (B) Modified size-exclusion chromatogram for 0.12 bmol each of a mixture of NTA, EDTA, and DTPA on a TSK-2000SW column. Conditions: eluent consists of buffer at pH 7.4 containing 10 ppm (1.6 X M) Cu(II), 0.10 M NaNO, and 0.05 M Tris at a flow rate of 0.5 mL min-'. The detector was the static dropping mercury drop (SDME) flow cell operated in a reverse-pulse amperometric mode.
Flgure 2. Modified size-exclusion chromatograms for 15 pL of 8 X M catechol using 10 ppm copper(I1) in the eluent on (A) TSK2000SW and (B) Synchropak GPC 100. Conditions for (A) is the same as Figure 1 whlle conditions for (B) are the same as (A) except the ionic strength was lowered by using 0.01 M NaNO, and 0.01 M Tris buffer at a flow rate of 0.3 mL min-'.
column was tested with 20-pL samples of 8 X M EDTA, which were injected as a function of flow rate. The number of theoretical plates increased gradually from a value of approximately 9800 plates/m t~ a value of approximately 13000 plates/m as the flow rate was incregsed from 0.3 to 0.7 mL/min. The efficiency then decreased slowly with increasing flow rate up to 1.1mL/min which produced approximately 10000 plates/m. A flow rate of 0.5 mL/min which had a HETP of 0.1 mm/plate was selected for most of the subsequent separations for peak area measurements. This represents a tenfold improvement in efficiency since a HETP of approximately 1mm/plate has been previously observed for the Sephadex gels (8, 16). The linearity of the detector response in terms of peak height to the amount of EDTA after separation on the TSK column was determined using concentrations from 2.5 X lo4 to 1.5 X M with an injection volume of 20 pL. The least-squares equation for four samples of EDTA between 5.0 and 30 nmoles was i (peak height in microamps) = (1.90 f 4.7 X (amount of EDTA, nanomoles) + (2.01 f 0.09) PA with syx = 0.090 PA. A statistical detection limit for the analysis of EDTA, which has been separated RS the copper complex via Hummel-Dreyer chromatography and detected electrochemicallyby using rpa, was calculated from the slope (m)and overall standard deviation(s) over the range of 5.0-30.0 nmoles. The detection limit = ts/m was calculated by using a t test at the 95% confidence level (17). A value of 0.119 nmol or 40 ng of EDTA was obtained as the detection limit with the above technique. The detector response was found to be particularly sensitive to flow pulsations. Similar limitations have been reported by other investigators,who have employed
electrochemical detectors for HPLC (18). Thus, an improved solvent delivery system or some method of signal processing (i.e., Fourier transform) (19)could be utilized to minimize the noise contribution and thereupon lower the detection limit. Other ligands which were tested on the TSK column included citric acid and catechol. The citric acid complex of copper was resolved at an unusually small elution volume (Ve/Vo 1.17), which is consistent with similar observations previously observed on Sephadex (8). An explanation is that copper(I1) has been shown to form dimeric complexes, Cu2L2, under the pH and concentration conditions employed in this study (20). Figure 2A illustrates a modified gel chromatogram obtained for catechol in the presence of copper(I1) on the TSK column. Unlike the Sephadex case where considerableadsorption was noted for the copper catechol complex, the chromatogram on TSK shows a peak ( Ve/Vo= 1.5) which is well resolved from the trough ( V e /Vo = 2.1). However, it should be noted that when pyrogallol, which contains a third hydroxy group, was injected into the column, a peak Ve/Vo= 1.97 was observed. The relatively large Ve/Vo for this Cu(I1) complex of pyrogallol vs. the trough ( Ve/Vo = 2.1) indicates partial retention of this complex by adsorption. Special precautions were taken to minimize air oxidation of pyrogallol solutions. All solutions were prepared from preboiled and nitrogen-deaerated water and stored under nitrogen. Solutions were used immediately after preparation or before 3-h time, whereupon discoloration occurred. Therefore, some adsorption of the copper(I1) catechol complex could be present on the TSK-column. Synchropak GPC 100 Column. The Synchropak GPC 100 column is a high-performance aqueous SEC column, which
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Flgure 3. Modified size-exciusion chromatograms on the Synchropak GPC 100 column with an eluent consisting of 10 ppm Cu(II), 0.10 M NaNO,, and 0.05 M Tris adjusted to pH 7.4 and at flow rate of 0.5 mL/min: (A) 135 pg of water-derived fulvic acid (WFA), (B) 130 pL of a natural water sample obtained from a slow-moving tributary (English Coulee) of the Red River, in Grand Forks, ND. The detector was as in Figure 1.
was examined in the modified Hummel-Dreyer mode. The column has been reported to consist of a glycerylpropylsilyl layer, which is covalently bonded to 10-pm porous spherical silica particles (21). Although the Synchropak GPC 100 column has been reported to adsorb lysozyme during separation (221, other proteins and peptides were not appreciably adsorbed on this column in another molecular weight study (23). In addition, the column has been shown to be effective in the separation of a number of water-soluble cellulosics with no evidence of non-size-exclusion modes if a high ionic strength buffer (0.7 M, pH 3.7) was utilized (24). Therefore, the GPC 100 column was tested to determine its applicability with the copper(I1)-fulvic acid system in the current study. Figure 2B is a chromatogram of catechol in the presence of copper(I1) on the GPC 100 column. The copper(I1)catechol complex peak is well resolved from the copper(I1) depletion trough because the ionic strength was relatively low (0.02). The peak area for the copper-catechol complex is within 5% of the trough area, which represents the copper depleted from the elution upon formation of the complex. The relatively small peak VelV , (1.2) at this low ionic strength is the partial result of ion-exclusion partitioning, which is characteristic of these types of columns used with ionic strengths less than 0.10 (25). Therefore, further studies on the fulvic acid complexes of copper(I1)were carried out at an ionic strength of equal to or greater than 0.10. Figure 3A is a modified SEC chromatogram of a water fulvic acid sample using 10 ppm copper(I1) in an eluent with an ionic strength of 0.15. A peak for the copper(I1)-fulvic acid complex
is noted at a retention volume, which is very close to the column exclusion volume under the experimental conditions. A shoulder is seen at a larger retention volume; however, both peaks are well resolved from the trough. There is an obvious difference in the total areas of the peak vs. the trough, which is the apparent result of a difference in the response of the amperometric detector to the copper-fulvic acid complex vs. uncomplexed copper(I1). Buffle and co-workers have extensively studied the electrochemistry of fulvic acids (26);they have shown that adsorption of fulvic acid on mercury electrodes is important in the presence and absence of metal ions (27). Therefore, the peak areas are probably enhanced for the copper(I1)-fulvic acid complexes due to increased current flow through the electrochemical cell as a result of these adsorption processes. Since the response of the detector toward the copper(II)-fulvic acid complex was complicated by apparent adsorption of the fulvic acid on mercury, the trough or copper(I1) depletion band was utilized in subsequent quantitation procedures. The linearity of the detector response (i.e., area of trough) to the amount of water fulvic acid (WFA) injected was determined using amounts of 5-30 pg with injection volumes of 5-30 p L of sample. The least-squares equation for a series of injections was A (area of trough in centimeters squared with 240 pC/cm2) = (56.0 f 1.7) (amount of WFA, milligrams) + (0.06 f 0.03) cm2. A statistical detection limit for water fulvic acid was calculated from the slope ( m )and overall standard deviation (s) of the above calibration curve by using the equation detection limit = t s / m with the t test at the 95% confidence level (17). A value of 1.2 pg of water fulvic acid was obtained with the above technique. The minimum concentration of water fulvic acid, which could be detected by the technique, is then 24 ppm when a 50-pL sample loop is utilized as described in the Experimental Section. The area of the trough was correlated with the depletion of metal ions when a strong, stoichiometriccomplexing agent, EDTA, was employed. A series of experiments were conducted to 2.0 X in which 4.0 X pmol EDTA was injected on the Synchropak column. Area measurements on this column also consistently produced a peak/trough ratio of unity. Thus, if one assumes 1:l copper/EDTA formation, the amount of depleted copper can be directly measured. The least-squares equation, which relates trough area to the amount of copper depleted, was A (area of trough in centimeters squared with 240 FC/cm2) = (160 f 5) (amount of copper depleted, micromoles) + (0.57 f 0.07) cm2, Utilization of this relationship and the above relationship allows for the direct measurement of the amount of water fulvic acid in a sample (milligrams) or the amount of copper, which complexes with the volume of water sample injected (micromoles/liter). Similar behavior for a soil-derived fulvic acid sample was observed on the SEC column; a peak for the copper(I1) complex was observed at the exclusion volume of the column with a shoulder prior to the metal depletion trough. An enhanced detector response was also noted for the soil fulvic acidcopper(I1) complex; therefore, trough measurements were used in calibration curves and detection limits. A calibration curve for a series of soil fulvic acid injections showed similar linearity as the WFA with calculated detection limit (tslm)of 1.3 pg. The above WFA and copper trough calibration curves were subsequently utilized to measure the complexation capacity of a local natural water sample. The sample was obtained from a slow-moving stream called the English Coulee, which runs through the University of North Dakota campus and is a tributary of the Red River of the North. Figure 3B shows a representative, modified gel permeation chromatogram for a natural water sample on the Synchropak column. A peak for
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copper(I1) complex is noted at the exclusion volume; however, two sharp troughs are observed at retention volumes which are significantly less than that observed for the "normal" depletion band of copper(I1). By comparison of Figure 3B with 3A, the third trough (at a retention time of 18.8 min or retention volume of 9.4 mL) is observed consistent with the depletion band of copper(I1) in the eluent. A second large peak at a retention volume of 10.0 mL (20.0-min retention time) with considerable tailing is observed just beyond the metal depletion trough. The placement and size of this peak is consistent with a complexing agent in the water sample which is adsorbed both on the column packing and the mercury electrode in the detector cell. Although the first two troughs could not be identified, the third trough or "normal" metal depletion band was utilized in an attempt to measure the complexing or binding ability of the water sample. Measurement of the area of the metal depletion trough resulted in a complexation capacity measurement of 15 x lo* mol L-l copper(I1) in water sample. Utilization of the calibration curve on the basis of the amount of fulvic acid results in a concentration of 45 mg L-l fulvic acid in the water sample. The above capacity value is reasonably consistent with complexation capacity measurements of other natural water samples, which typically are (1-20) X lo4 mol L-l(28). Thus, the Synchropak GPC 100 column appears to be potentially useful in the measurements of binding agenta in water systems when the column is utilized in a Hummel-Dreyer mode with excess metal ions in the eluent.
ACKNOWLEDGMENT We thank James Weber, University of New Hampshire, for the soil-derived fulvic acid sample used in this study. Registry No. Spherogel TSK-2000 SW, 93348-96-0; Synchropak GPC 100, 93348-97-1;water, 7732-18-5.
LITERATURE CITED (1) (2) (3) (4) (5) (6)
(7) (8) (9) (10)
(11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28)
Barth, H. G. J . Chromatogr. Sci. 1983, 78, 409-429. Dubin, P. L. Am. Lab. (Fairfield, Conn.) 1983, 75,62-73. Salto, Y.; Hayano, S. J . Chromatogr. 1979, 777, 390-392. Miles, C. J.; Brezonik, P. L. J . Chromatogr. 1983, 259, 499-503. Swift, R. S.; Posner, A. M. J . SoilSci. 1971, 2 2 , 237-249. Rashld, M. A.; King, L. H. Geochim. Cosmochim. Acta 1969, 33, 147-151. GJessing,E. T. Nature (London) 1985, 208, 1091-1092. Adarnlc. M. L.; Bartak, D. E. Anal. Chlm. Acta 1984, 158, 43-55. Maitoza, P.; Johnson, D. C. Anal. Chim. Acta 1980, 778, 233-241. Weber, J. H.; Wilson, S. A. Water Res. 1975, 9, 1079-1084. Kato, Y.; Komlya, K.: Sasakl, H.: Hashlmoto, T. J . Chromafogr, 1980, 100, 297-303. Rokushika, S.; Ohkawa, T.; Hatano, H. J . Chromatogr. 1979, 776, 456-461. Wehr, C. T.; Abbott, S. R. J . Chromatogr. 1979, 785, 453-462. Yoza, N. J . Chem. Educ. 1977, 5 4 , 284-287 and references thereln. Mantoura, R. F. C.;Riley, J. P. Anal. Chlm. Acta 1975, 78, 193-200. Sosa, J. M. Anal. Chern. 1980, 52, 910-912. Skogerboe, R. K.; Grant, C. L. Spectrosc. Lett. 1970, 3 , 215-220. Weber, S. 0.; Purdy, W. C. Anal. Chlm. Acta 1978, 700, 531-544. Weber, S. G. Anal. Chem. 1982, 5 4 , 2126-2127. Still, E. R.; Wikberg, P. Inorg. Chim. Acta 1980, 46, 147-152. Regnier, F. E.; Noel, R. J . Chromatogr. Sci. 1976, 74, 316-320. Pfannkoch, E.; Lu, K. C.; Regnler, F. E.; Barth, H. G. J . Chromatogr. SCi. 1980, 18, 430-441. Gruber, K. A.; Whitaker, J. M.; Morrls, M. Anal. 8iochem. 1979, 9 7 , 176-1 63. Barth, H. 0.; Regnier, F. E. J . Chromatogr. 1980, 192, 275-293. Kopaciewicz, W.; Regnler, F. E. Anal. 8iOChem. 1982, 726, 8-16. Cominoli, A.; Buffie, J.; Haerdi, W. J . Hectroanal. Chem. 1980, 770, 259-275. Buffle, J.; Greter, F. L. J . flectroanal. Chem. 1979, 707, 231-251. Sear, R. A.; Weber, J. H. Environ. Sci. Techno/. 1982, 76, 510A517A.
RECEIVED for review May 29, 1984. Accepted September 14, 1984. This work was supported by matching Grant B-057NDAK from the Office of Water Research and Technology, administered by the North Dakota Water Resources Research Institute.
Size-Exclusion Chromatography and Size-Exclusion Chromatography/Fourier Transform Infrared Spectrometry of Reacting Organofunctional Trialkoxysilanes James D. Miller and Hatsuo Ishida* Department of Macromolecular Science, Case Western Reserve University, Cleveland, Ohio 44106
Slre-excluslon chromatography (SEC), severally and in combination wlth Fourier transform Infrared (FT-IR) spectroscopy, is shown to be unlquely appllcable to the lnvestlgatlon of the hydrolysis and condensatlon reactions of organofunctlonal trlaikoxysllanes. Sire-exclusion chromatography provides a method of separation whlch reveals previously undemonstrated lnformatlon about the reactlons of trlaikoxysllanes. Interfaclng the chromatography to a FT-IR spectrophotometer wlth dlffuse reflectance optlcs allows identlflcatlon of Individual reactlon products Including the silanetriol and the cyclic tetramer. Reaction kinetics are followed for phenyltrlethoxysilane In aqueous tetrahydrofuran. The behavlor of the reactlng slianes In different media (e.g., aqueous and aqueous-organic) can also be monltored and Is demonstrated to be substantlally different.
Most analytical techniques have been applied to organofunctional silanes in an attempt to explain their beneficial and
sometimes mysterious behavior in a wide variety of applications (e.g., reinforced polymer composites, liquid crystal orientation, and enzyme immobilization). The reactions of organofunctionaltrialkoxysilanes are numerous, complex, and depend on a number of interacting variables. Therefore, the investigation of these systems has required the use of many and varied techniques in order to obtain even a preliminary picture of the chemistries involved. If there is a major class of analysis which has been relatively neglected throughout the 30 or more years of study in this field it would have to be chromatography. Only a few citings are available in which any type of chromatographic separations are used to analyze organofunctional trialkoxysilanes. Plueddemann ( I ) employed gas chromatography (GC) to separate the lower members of trimethylsilyl end-blocked siloxanes formed from condensing vinylsiloxanols and vinyl siliconates by the Lentz (2)technique. GC has also been used to determine whether a silane adsorbs onto a substrate surface from solution (3). Thin-layer chromatography (TLC), using Rhodamine B dye or Bromocresol Green as developing agents for
0003-2700/85/0357-0283$01.50/00 1984 American Cbernical Society
