Anal. Chem. 1994,66, 2841-2851
Sulfur-Selective Detector for Liquid Chromatography Based on Sulfur Monoxide-Ozone Chemiluminescence Thomas B. Ryerson, Andrew J. Dunham, Robert M. Barkley, and Robert E. Sievers' Department of Chemistry and Biochemistry, CIRES, and Global Change and Environmental Quality Program, University of Colorado, Boulder, Colorado 80309
We report here a new detector for HPLC, employing gasphase chemiluminescence for selectivedetectionof liquid-phase sulfur compounds. This detector operates by converting sulfurcontaining compounds in the liquid phase, under pressure and at elevated temperatures, into sulfur monoxide. The SO is allowed to permeate across a membrane, into a helium stream, and is swept into a reaction cell. Ozone is added to the cell, and photoemission resulting from the SO 0 3 reaction is monitored by a photomultiplier tube. The method reported here has been shown to operate effectively at 0.4-9.9 mL/min flow rates, typical of analytical HPLC, ion chromatography, and flow injection analysis. The design and performance of the new liquid-phase sulfur analyzer is described. The parameters most affecting the detector response are the reaction capillary temperature and pressure, the composition of the liquid mobile phase, and the oxidation state of sulfur in the parent analyte. Application of the detector to reversed-phase HPLC analysis of standards of sulfur-containing pesticides, proteins, and blood thiols and FIA of acid-soluble thiols in rat plasma is presented.
+
Analysis of sulfur compounds in the complex matrices typical of environmental or biological samples presents two distinct problems: separation and identification. Separation requires the analyte be isolated from other species in the matrix that are analytically less significant. Chromatographic separation methods are commonly employed, often in combination with less selective separations such as filtration, extraction, and/or digestion. For many analyses, sample cleanup and chromatography are sufficient to separate the sulfur-containing analytes from each other and from other compounds in the sample matrix. However, for more complex samples common in environmental and biological analyses, the resolving power of modern chromatographic techniques may still be insufficient to fully isolate all the sulfur-containing compounds of interest. The recent trend has been to couple selective detectors with separation processes for complex analyses. A selective detector can alleviate both problems, separation and identification, by only responding to the analyte of interest regardless of whether other constituents of the matrix remain unseparated from the analyte. Some sulfur compounds can be detected directly by UV absorbance at 254 nm.' However, because so many other organic compounds also absorb at that wavelength, this cannot be considered a selective detection method for sulfur. Both (1) Chun, B.-G.; Paik, W. K.; Kim, S.J . Chromatogr. 1983, 264, 321-328.
0003-2700/94/0366-2841$04.50/0 0 1994 American Chemical Society
precolumn derivatization2and postcolumn derivatizatiod have been employed to label sulfur compounds with a chromophore for subsequent UV absorbance or fluorescence detection. Electrochemical methods for liquid-phase sulfur detection have been reported and involve redox chemistry of sulfur compounds4 and of sulfur compound photolysis product^.^ Other spectroscopic methods used for sulfur-selective detection in HPLC include liquid-phase chemiluminescence,6 inductively coupled plasma atomic emission ~pectrometry,~ inductively coupled plasma mass spectrometry,8 and flame photometry.9 Luminescence methods have also been used, based on indirect detection of sulfur compounds by quenching of 3-aminofluoranthene6 or biacetyl.1° These latter methods are sensitive to reduced sulfur compounds only. Although chemiluminescence (CL) detectors for liquid chromatography are relatively new, a variety of C L schemes have been reported re~ent1y.Il-I~Two approaches require nebulization of the entire LC effluent directly into the evacuated reaction chamber of a chemiluminescence detector, where analyte sulfur-containing compounds are reacted with molecular fluorine14 or with ozone.I6 Sulfur-selective chemiluminescence detection can be achieved by converting sulfur-containing compounds to sulfur monoxide and subsequently allowing the SO to react with ozone to produce electronically excited S02*:17
Conversion to SO prior to ozone oxidation results in enhancement of the CL response from sulfur compounds by up to 3 (2) Ogasawara, Y.; Ishii, K.; Tagawa, T.; Tanabe, S.Analysr 1991,116, 13591363. (3) Obrezkov,O. N.;Shpigun,O. A.; Zolotov, Y. A.;Shlyamin, V. I.J.Chromatogr. 1991, 558, 209-213. (4) Vandeberg, P. J.; Johnson, D. C. Anal. Chem. 1993, 65, 2713-2718. (5) Dou, L.; Krull, I. S.Anal. Chem. 1990, 62, 2599-2606. (6) van Zoonen, P.; Bock, H.; Gooijer, C.; Velthorst, N. H.; Frei, R. W.Anal. Chim. Acra 1987, 200, 131-141. (7) Biggs, W. R.; Fetzer, J. C. Anal. Chem. 1989, 61, 236-240. (8) Jiang, S.-J.; Houk, R. S.Spectrochim. Acra 1988, 438, 415-421. (9) Julin, B. G.; Vandenborn, H. W.; Kirkland, J . J. J . Chromarogr. 1975, 112, 443-453.
(IO) Donkerbroek, J. J.; Veltkamp, A. C.; Gooijer, C.; Velthorst, N. H.;Frei, R. W. Anal. Chem. 1983, 55, 1886-1893. (11) Nelson, J . K.; Getty, R. L.; Birks, J. W. Anal. Chem. 1983.55, 1767-1770. (12) Shearer, R. L.; ONeal, D. L.; Rios, R.; Baker, M. D. J . Chromatogr. Sci. 1990, 28, 2 4 2 8 . (13) Chang, H.-C. K.; Taylor, L. T. Anal. Chem. 1991, 63, 486-490. (14) Mishalanie, E. A.; Birks, J. W . Anal. Chem. 1986, 58, 918-923. (15) Shearer, R. L. Anal. Chem. 1992, 64, 2192-2196. (16) Birks, J. W.; Kuge, M. C . Anal. Chem. 1980, 52, 897-901. (17) Benner, R. L.; Stedman, D. H. Anal. Chem. 1989, 61, 1268-1271.
AnalyticalChemistry, Vol. 66, No. 18, September 15, 1994 2841
+
orders of magnitude, relative to direct reaction of the precursor flow rates of HPLC. In order to apply SO O3 chemiluorganosulfur compound with ozone. minescence to analytical-scale HPLC, two difficulties must be overcome: efficient postcolumn production of SO and rapid There are several means of producing SO from sulfurtransfer of the SO to a chemiluminescence cell. Because SO containing compounds: by passing certain sulfur compounds is a thermodynamically unstable diradical, production of free through a plasma,'* through an electrical discharge,Ig by SO usually requires high-energy processes;38furthermore, once thermal decomposition/pyrolysis of hafnium or aluminum sulfates, sulfites, and dithionates,20*21of thiirane o ~ i d e s , ~ ~ ,formed, ~ ~ the SO must be allowed to react rapidly with ozone for detection before undesirable side reactions consume it. In or of thionyl halides,24and by combustion of sulfur-containing the present study, we report the development of a sensitive compound^.^^^'^ Of these methods, until now only combustion sulfur-selective detector that can be used at liquid flow rates has been shown to be of practical use in chromatographic (ca. 1 mL/min) characteristic of analytical-scale HPLC, ion S-compound detection. Two sulfur-selective detectors based chromatography (IC), and flow injection analysis (FIA). on combustion followed by SO + O3 CL are available. One, termed the flame sulfur chemiluminescence detector (SCD),25 has been shown to exhibit nearly equimolar response to sulfur EXPER I MENTAL SECT1ON compounds regardless of oxidation state.I2 Under optimal Reagents. Doubly distilled deionized water was degassed flame conditions, no significant interference from coeluting by vacuum aspiration and continuously sparged with highcompounds is noted.26 Selectivity over hydrocarbons is greater purity helium before use as mobile phase and reagent. than 107.12Flame SCD shows a linear range of 5 orders of Potassium iodide, 30% hydrogen peroxide, potassium nitrate, magnitude, and detection limits have been reported as low as dimethyl sulfoxide, sodium sulfite, and concentrated hydro0.4 pg of S/s when the conditions were fully optimized. A chloric, sulfuric, and phosphoric acids were used as received second combustion method for SO formation has been reported from Mallinckrodt Specialty Chemicals Co. (Chesterfield, very recently. 5,27 MO) . L-cysteine,m-cystine, N-acetyl-L-cysteine, glutathione The combination of high sensitivity and selectivity, wide (GSH), oxidized glutathione (GSSG), DL-methionine, glutamlinear range, and lack of quenching and interferences from ic acid, caffeine, sodium nitrite, penicillamine, and penicilnon-sulfur species has made flame SCD an effective choice lamine disulfide were used as received from Aldrich Chemical for chromatographic detection of sulfur compounds. ChroCo., Inc. (Milwaukee, WI). Dithiothreitol was obtained from matographic analyses of sulfur g a s e ~ , ~ ~PTH-amino -~O acids,I3 Boehringer Mannheim Corp. (Indianapolis, IN). N-Methn-alkanesulfonic acids,13sulfur-containing pesti~ides,l3,~~,3~-33 ylcarbamate standards were purchased from Alltech Associpolycyclic aromatic sulfur-containing hydrocarbons,32nonionic ates (Deerfield, IL). Urea, sodium sulfate, and iron(I1) surfactant^,^^ and sulfur compounds in beer,34natural gas,35 chloride tetrahydrate were purchased from Fisher Scientific diesel and petroleum using this method Co. (Fair Lawn, NJ). Dimethylthiourea was obtained from have been reported. This design has been applied to gas Alfa/Johnson-Matthey (Ward Hill, MA). Disodium EDTA chromatography (GC),12326329J4-36 supercritical fluid chrodihydrate was purchased from J. T. Baker Inc. (Philipsburg, matography (SFC),31and microscale capillary liquid chroNJ). Thiirane 1-oxide was synthesized in our laboratory by matography (LC).I3J3 However, no analogous method of the method of Hartzell and Paige.23 Oxygen, air, and highflame conversion exists for analytical-scale high-performance purity helium were purchased from U.S. Welding Inc. liquid chromatography (HPLC), presumably due to the (Boulder, CO). Finally, sulfur dioxide, hydrogen sulfide, and difficulty of interfacing a flame with the relatively high liquid nitrogen dioxide were obtained from Matheson Gas Products (Denver, CO). (18) Halstead, C. J.; Thrush, B. A. Photochem. Photobiol. 1965, 4, 1007-1013. (19) Cordes, V. H.; Schenk, P. W. Z . Anorg. Allg. Chem. 1933, 214, 3 3 4 3 . Apparatus. A Model 1330 HPLC pump (Bio-Rad Labo(20) Papazian, H. A.; Pizzolato, P. J.; Orrell, R. R. Thermochim. Acta 1972, 4, ratories, Hercules, CA) was used to pressurize and deliver the 97-103. ( 2 1 ) Papazian, H. A.; Plzzolato, P. J.; Peng, J. Thermochim. Acta 1972, 5 , 147reagent/mobile phasein FIA and isocratic HPLC separations. 152. A Model 9010 solvent delivery system (Varian Analytical (22) Aalbersberg, W. G. L.; Vollhardt, P. C. J . Am. Chem. SOC.1977, 99, 27922794. Instruments, San Fernando, CA) was used for gradient HPLC (23) Hartzell, G. E.; Paige, J . N. J . Am. Chem. SOC.1966.88, 2616-2617. separations. Injections were made with a Model 7 125 manual (24) Schenk, P. W.; Blwhing, H. Chem. Ber. 1959, 92, 2333-2337. (25) SCD is a registered trademark issued to Sievers Instrument Co., 2500 Central injection valve (Rheodyne Inc., Cotati, CA) equipped with a Ave., Boulder, CO. fixed-volume injection loop. A heating block was machined (26) Johansen, N. G.; Hutte, R. S.;Legier, M. F. Monitoring Water in the 1990's: Meeting New Challenges; Hall, J. R., Glysson, G. D., Eds.; ASTM: out of two 8.0-in.-long aluminum blocks bolted together and Philadelphia, PA, 1991. heated with two 400-W cartridge heaters (CIR-2047/ 120, (27) Shearer, R. L.; Poole, E. B.; Nowalk, J . B. J . Chromatogr. Sci. 1993, 31, 82-87. Omega Engineering, Stamford, CT) connected to an Omega (28) Benner, R. L.; Stedman, D. H. Enuiron. Sci. Technol. 1990, 24, 1592-1596. CN2400A temperature controller driving a 25-A solid-state (29) Hutte. R. S.;Johansen, N. G.; Legier, M. F. J . High Resolut. Chromatogr. 1990, 13, 421-426. relay. This heater allowed thermostating of the reaction (30) Gaines, K. K.; Chatham, W. H.; Farwell, S. 0.J . High Resolut. Chromatogr. capillary at temperatures from ambient to 450 f 2 "C. Various 1990, 13, 489493. (31) Chang, H.-C. K.; Taylor, L. T. J . Chromafogr. 1990, 517, 491-501. materials were used for constructing the capillary reaction (32) Howard, A. L.; Taylor, L. T. Anal. Chem. 1993, 65, 7 2 4 1 2 9 . tubes. Nickel capillary and aluminum tubing were both (33) Howard, A. L.; Thomas, C. L. B.; Taylor, L. T. Anal. Chem. 1994,66, 14321437. obtained from Small Parts, Inc. (Miami Lakes, FL), fused (34) Burmeister, M. S.;Drummond, C. J.; Pfisterer, E. A,; Hysert, D. W.; Sin, Y. silica-lined stainless steel (SS) tubing was from Restek Corp. 0.;Sime, K. J.; Hawthorne, D. B. J . A m . SOC.Brew. Chem. 1992,50,53-58. (35) Johansen, N. G.; Birks, J. W. Amer. Lab. 1991, 23, 112-119. (Bellefonte, PA), ceramic alumina tubing was from Coors (36) Chawla, B.; Di Sanzo, F. J . Chromatogr. 1992, 589, 271-279. (37) Eckert-Tilotta, S. E.; Hawthorne, S. B.; Miller, D. J. J . Chromatogr. 1992, 591, 313-323.
2042
Analytical Chemistry, Vol. 66, No. 18, September 15, 1994
(38) Schenk, P. W.; Steudel, R. Angew. Chem., Int. Ed. Engl. 1965.4, 402-409.
Mobile phase reservoir
tqLV;
He sweep gas in I
Permeation cell
Dl
Ozone
generator
,A
Integrator
I'
t HPLC column '
Reaction capillary
Liquid Cooling bath
waste
u SCD 350A (with no flame) a. CL cell b. optical filter
Figure 1. Schematic of the liquid-phase sulfur analyzer.
Ceramics (Golden, CO), and stainless steel capillary tubing was obtained from Alltech. A Rheodyne Model 7037 pressure relief valve operated in the constant back-pressure mode was used to establish back pressure independently of the liquid flow rate in the reaction capillary. Two different permeation cells were designed and constructed and are described in detail below. Microporous Gore-tex tubing was purchased from Anspec Co., Inc. (Ann Arbor, MI), and Teflon tape was obtained from Swagelok Co. (Solon, OH). The chemiluminescence detector was a SCD 350A (Sievers Instruments, Inc., Boulder, CO). TheSCD was connected to the permeation cell by a 5-ft length of opaque poly(tetrafluoroethy1ene) (PTFE) transfer line provided with the SCD. Mass spectrometric experiments were carried out using a VG 7070 instrument (Fisons Instruments, San Carlos, CA) with EBQQ geometry. A 5890 Series I1 G C flame ionization detector (Hewlett-Packard Co., Palo Alto, CA) was used for the flame conversion experiments described in the text. Sample Preparation: Rat Plasma. Blood samples were obtained from two male Sprague-Dawley rats and centrifuged to separate cells from plasma. The plasma was treated with perchloric acid to precipitate any proteins and centrifuged at 10 000 rpm for 15 min. Theclear supernatantwas thendrawn off and filtered through a filter disk with 2-pm pore size, and three 20-pL aliquots from each rat were analyzed. NO2 Permeation Cell. A permeation cell was constructed and calibrated to supply a known amount of N O Z ( ~for ) the gas-phase titration of SO. NO2 from a lecture bottle was cooled and condensed into a test tube held at 0 "C by immersion in an ice-water bath. Pure oxygen was bubbled through the condensed liquid for 15 min to oxidize any trace N O to NO*. Purified NO20) was decanted from the test tube into a 6-in.long, f 4-in.-0.d. piece of Teflon tubing, and the tubing ends were then sealed. The sealed tube was placed in a flow cell immersed in a constant-temperature water bath, and a constant stream of helium directed through the cell. The concentration of NO2 in the helium stream was determined by measuring the mass of NO2 lost over time through the pores of the cell and measuring the flow rate of helium through the cell.
RESULTS AND DISCUSSION In order to extend the advantages of SO + 03 C L detection to HPLC, we sought an alternative method for formation of SO from dissolved sulfur compounds that did not require a flame. The research focused on pyrolysis, hydrolysis, and
partial oxidation in the liquid phase. Other workers have studied the wet air oxidation and supercritical water (SCW) oxidation of organic compounds, but there was no indication of whether SO might be formed as a product from sulfurcontaining compounds.3g45 The new liquid-phase sulfur analyzer (LPSA) that we have developed consists of the following components: a reagent/ mobile-phase reservoir, a high-pressure liquid pump, an injection valve, an analytical HPLC column, a heated capillary reaction chamber, a cooling bath, a flow restrictor, a permeation cell, and a sulfur chemiluminescence detector consisting of an ozone generator, an evacuated C L chamber, a photomultiplier tube, and a vacuum pump (Figure 1). (The configuration for HPLC is shown in this figure; for FIA experiments, the analytical column was omitted). Sulfur compounds injected into the liquid stream are pumped through a heated capillary reaction tube, which is maintained under pressure to prevent vaporization of the mobile phase and maintain the stream in a liquid state. Conversion of sulfur compounds to SO takes place under moderately high temperature and pressure (e.g., 300 OC and 2000 psig) in the reaction capillary. Downstream of the capillary, the liquid stream is cooled by means of a cooling bath and the pressure reduced to ambient by means of a flow restrictor. Dissolved gases are subsequently allowed to permeate from the liquid phase into the gas phase across a hydrophobic, microporous membrane (Figure 2). Permeant gases are then withdrawn under vacuum and allowed to react with ozone, and the resulting emission is measured and quantitated. Optimization. Optimization of the LPSA response to liquid-phase sulfur compounds can be thought of as divided into two general operational areas. The first area is optimization of the liquid-phase conversion process by which SO is formed from the sulfur-containing analyte. LPSA response for a given sulfur compound is most dependent on reaction temperature, but several other variables were studied, including (39) Chang, K.-C.; Li, L.; Gloyna, E. F. J. Hazard. Mater. 1993, 33, 51-62. (40) Helling, R. K.; Tester, J. W. Enuiron. Sci. Techno/. 1988, 22, 1319-1324. (41) Huppert, G. L.; Wu, B. C.; Townsend, S.H.; Klein, M. T.; Paspek, S.C . Ind. Eng. Chem. Res. 1989, 28, 161-165. (42) Jin, L.; Ding, 2.;Abraham, M. A. Chem. Eng. Sei. 1992, 47, 2659-2664. (43) Mok, W.S.-L.; Antal, M. J. J.; Jones, M. J. J. Org. Chem. 1989, 54,45964602. (44) Ramayya, S.;Brittain, S.;DeAlmeida, C.; Mik, W.; Antal, M.J. J. Fuel 1987, 66, 13641371. (45) Townsend, S.H.; Abraham, M. A.; Huppert, G.L.; Klein, M.T.; Paspek, S. C . Ind. Eng. Chem. Res. 1988, 27, 143-149.
Analytlcal Chemistw, Vol. 66, No. IS, September 15, 1994
2843
ToCL cell
Hesweep gas in
1.2 IO'
,
"
'
I
"
'
,
"
'
,
~
"
1.0 IO'
8.0 IO6
6.0IO6
I
1I-
I
microporous membrane '(e.g., Teflon tape)
2.0 106
1
1 I
Il
' ' . + ' =
00
0
I
t
1
T o liquid waste
Figure 2. Schematic of the flat-membrane permeation cell. Upper and lower pieces are constructed of stainless steel. The permeation membrane is sandwiched betweenthe upper and lower pieces, forming a liquid-tight seal. Liquid stream fittings are modified 1/16-in.SS lowdead-volume fittings, silver-brazed into permeation cell body. Total internal liquid-stream volume is less than 100 pL.
liquid flow rate and back pressure, reagent/mobile-phase composition and pH, and the material of construction of the capillary reaction chamber. The second area of optimization is the permeation process, involving transfer of dissolved SO to the gas phase. Variables in this process include permeation cell design, type of permeation membrane used, and sweep gas pressure. A discussion of the variables in LPSA operation and their optimization for FIA is presented below. Optimization specific to HPLC is discussed in a following section. Liquid-Phase Conversion Optimization: Reaction Temperature. A typical plot of temperature versus response for a sulfur-containing model analyte is shown in Figure 3. This analyte, dimethylthiourea (DMTU), was chosen on the basis of its nearly identical behavior to sulfur-containing proteins and amino acids in the new detector system, its purity, and its ready solubilityin water. A minimum temperature of 180 "Cis required to initiate the conversion chemistry. A response maximum is reached between 280 and 300 "C. At higher temperatures, response varies over a range of 20% until the temperature limit of the heater is reached. We believe that the occurrence of a maximum near 300 "C is a result of two competing reactions, the first forming SO and the second destroying it (presumably by further oxidation to S02, which does not chemiluminesce upon oxidation with ozone in the gas phase): S-compound(,,) SO,,,, + products (la)
-
-
-
SO is a relatively unstable diradical and is easily oxidized in air to the more stable dioxide,38 thus reaction l b is both thermodynamically favored and kinetically fast. Therefore, the reaction temperature must be sufficient to form SO via reaction l a but not high enough that reaction l b becomes significant. 2844
I
200
.
300
.
.
I
400
.
.A 500
Temperature, "C
I
Liquid sample in
100
AnalyticalChemistry, Vol. 66, No. 18, September 15, 1994
Figure 3. Typical temperature response curve for dissolved sulfur compounds. Responses, at each temperature, from five 20-pL injections of 500 pM DMTU were averaged; plotted are averages f 1 standard deviation. FIA conditions were as follows: 1.0 mVmin flow rate at 3300 psig distilled water, continuously sparged in the mobile-phase reservoir with helium; upper trace is response generated with mobile phaseadjusted to pH = 2.3 with H3P04,while the lower trace is response obtained with no added acid. The reactor capillary was '/le in. 0.d. by 0.020 in. i.d. stainless steel, 24 in. long, of which 8 in. was heated. Flat-membrane cell with Teflon tape membrane: CL cell maintained at 10-Torr pressure.
Sensitive responses to sulfur-containing compounds in the LPSA have been obtained under conditions at which water was in a supercritical state. However, for two reasons, most of the present work was done under less extreme conditions of temperature and pressure. First, supercritical water is an extremely corrosive medium. This corrosivity and the higher pressure impose considerable stresses on the pump, reactor tubing, back-pressure valve, and associated fittings. Second, at higher reaction capillary temperatures, the back pressure must be increased to prevent vaporization of the liquid stream. Therefore, when used as a HPLC detector, the total system pressure is relatively high, because the back pressure required in the reactor is added to the back pressure afforded by the analytical column. Increased pressure on the analytical column packing translates into decreased column lifetime. We have chosen 280-300 "C as the optimum temperature range, because small temperature deviations in this range result in the smallest changes in response. In practice, the deviations in reaction temperature are within k 2 "C, and responses from repetitive injections of 3 pM sulfur concentration are reproducible within go%) in the CL signal from injections of 10pM DMTU was observed when trace amounts of NO2 were added to the sweep gas stream of the new detector. Mass spectrometric experiments, along with results from thiirane 1-oxide injections and gas-phase NO2 titration, have provided evidence for SO as the species formed and detected by aqueous-phase conversion followed by ozone-induced chemiluminescence. These findings imply considerably greater stability of SO in the aqueous phase than has previously been recognized. In summary, the optimum conditions for FIA analysis of dissolved sulfur compounds are presented in Table 1. Similar conditions are expected to be optimal for HPLC and ion chromatography; the effects of changes in HPLC mobilephase composition are discussed below. Once optimum conditions for CL detection of dissolved sulfur-containing compounds were determined, the sensitivity and selectivity of this method was evaluated. The results are presented below. Sensitivity. A wide range of dissolved sulfur compounds can be sensitively detected in the LPSA. Sulfur-containing pesticides, proteins, and amino acids, as well as inorganic sulfur compounds, have been detected at sub-ppm levels. Response to sulfur compounds, under the conditions listed in Table 1, ~~
Repetitive injections of thiirane 1-oxide solutions produced 2040
AnaiyticalChemistry, Vol. 66,No. 18, September 15, 1994
(47) Black, G ,Sharpless, R L , Slanger. T G Chem Phys Lett 1982,90,55-58
s1
Table 1. Recommended Operatlng Parameters for Analysls of Dissolved Sulfur-Containlng Compounds mobile phase/reaction medium mobile-phase pH mobile-phase flow rate heater temperature back pressure in reaction chamber reaction capillary permeation cell design, membrane permeation cell sweep gas, pressure CL cell pressure
s2
helium-sparged water adjusted to pH lo’ Yes GC, SFC, p-HPLC
3 none reported
>IO* Yes GC, SFC
~~
LPSA (this work)
16* 3 3 e l u t i n g compds at >10 wt % NO,-, C& >I@ >I@/ no no GC HPLC 2d
alcohols
4 As thiophene. As thiophene. As 3-methylthiophene. As 2,5-dimethylthiophene. As dimethylthiourea. /Selectivity determined vs nitrate ion and urea.
HPLC. This detector complements the flame SCD, which has been successfully interfaced with GC, SFC, and microscale HPLC. Potential applications of this new detector in HPLC include analyses of complex biological fluids for sulfurcontaining proteins, thiols, pharmaceuticals, and their metabolites and analyses of natural waters for reduced sulfur species such as dimethyl sulfide. A brief summary of chromatographic detection limits and characteristics of several SO + O3-based detection schemes is presented in Table 3, with a comparison to the presently reported method.
of liquid-phase modifiers, need to be examined as new systems arestudied. Thedetector exhibits a linear responseof 3 orders of magnitude of analyte concentrations. A selectivity for detection of sulfur-containing compounds, over most nonsulfur-containing species, of greater than lo6 was observed. Detection limits for reduced sulfur-containing species, such as blood thiols, peptides, and proteins, is on the order of 10 ppb S in aqueous solutions. Other compounds that can be detected include various sulfur-containing pesticides and pharmaceuticals, as well as inorganic sulfur species.
CONCLUSIONS We have described the design, operation, performance, and representative applications of a new sulfur-selective chemiluminescence detector for HPLC and flow injection analysis. Sulfur monoxide is formed from dissolved sulfur compounds at elevated temperature and pressure; after permeation across a membrane to the gas phase, the chemiluminescent SO + 03 reaction is used to quantitate the analyte selectively. Response dependence on temperature and liquidphase pH and composition was studied. Capillary reaction chamber surface effects, and the presence of differing amounts
ACKNOWLEDGMENT Support from the N S F under Grant ATM-9115295 is gratefully acknowledged. We thank Sievers Instruments Co. for the generous gift of a sulfur chemiluminescence detector. Dr. Brooks Hybertson of the Webb-Waring Lung Institute kindly provided the rat plasma samples, and Dr. Richard Baltisberger provided us with helpful suggestions and comments during the development of the detector. We also thank C. Orr and G. Wiegand for assistance in developing the HPLC separations. Kris Hansen provided invaluable assistance in the preparation of the manuscript.
(49) Gaffney, J. S.; Spandau, D. J.; Kelly, T. J.; Tanner, R. L. J . Chromatogr. 1985, 347, 121-127. (50) Ryerson, T. B.; Barkley, R. M.; Sievers, R . E. J . Chromafogr.,in press.
Received for review January 4, 1994. Accepted M a y 26, 1994.’ e Abstract published
in Aduance ACS Absfracfs,July IS, 1994.
AnalyticalChemistty, Vol. 66, No. 18, September 15, 1994
2851
