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provide for greater sensitivity and a longer lived ion current. CONCLUSIONS The two types of discrimination that occur in quantitative fatty acid mixture analysis by FAB mass spectrometry depend on the concentration regime of the analysis. By considering the origin of this discrimination, the requirements of an improved FAB matrix were determined. Dodecanol meek these requirements, a t least for mixtures of saturated fatty acid congeners. However, the lack of surface activity and the more volatile nature of this matrix make it less compatible with MS/MS than the typical FAB matrices. We are currently investigating the use of dodecanol in the analysis of mixtures containing more widely different fatty acids, as well as the possibility of quantification without the use of an internal standard. The application of the basic principles utilized in this study is being considered for other analyte systems.
Ligon, W. V.; Dorn, S. B. Int. J . Mass Spectrom. Ion Processes 1984. 6 1 , 3113-122. Lyon, P. A.; Hunt, S. Presented at the 36th ASMS Conference on Mass Spectrometry and Allied Topics, 1988. Naylor, S.; Findeis, F. A.; Gibson, B. A.; Williams, D. H. J . Am. Chem . SOC. 1988, 108, 6359-6362. Bull, H. 6.; Breese, K. Arch. Biochem. Bbphys. 1974, 161, 865. Caprioii, R. M.; Morre, W. T.; Fan, T. Rapid Commun. Mass Spectrom. 1987, 1 , 15-18. Jensen, N. J.; Tomer, K. 6.; Gross, M. L. Anal. Chem. 1985, 5 7 , 2018. Bull, H. B. An Introdwtlon to physical Bbchemisby; F. A. Davis Company: Philadelphia, PA, 1964; Chapter 9. Naylor, S.; Skelton. N. J.; Williams, D. H. J . Chem. Soc., Chem. Commun. 1986, 1619. Deterdlng, L. J.; Gross, M. L. Anal. Chlm. Acta 1987, 200, 431. Adams, J.; Gross, M. L. Org. Mass Spectrom. 1988, 2 3 , 307. Gross, M. L.; Chess, E. K.; Lyon, P. A.; Crow, F. W.; Evans, S.; Tudge. H. Int. J. Mass Spectrom. Ion phys. 1982. 42, 243. Barber, M.; BordoH, R. S.; Ellott, 0. J.; Sedgwick. R. D.; Tyler, A. N. J . Chem. Soc., Faraday Trans. 11983. 79, 1249-1255. Mead, J. F.; Alfin-Slater, R. B.; Hewtan. D. R.; Popjak, G. LIPIDS Chemisby, Blochemlstry, and Nutrnotion; Plenum Press: New York, 1986; Chapter 4.
LITERATURE CITED Barber, M.; Bordoli, R. S.; Elliott, G. J.; Sedgwick, R. D.; Tyler, A. N. Anal. Chem. 1982, 5 4 , 645A. Bleman, K.; Martin, S. A. Mass Specfrom. Rev. 1987. 6. 1. Bleman, K.; Scoble, H. A. ScEence 1987, 237, 992. Hunt. F. D.; Yates, J. R. III; Shabanowk, J.; Winston, s.; buer, C. R. Proc. Natl. Acad. Sei. USA 1986, 8 3 , 6233-6237. Tomer, K. 8.; Jensen, N. J.; Gross, M. L. Anal. Chem. 1988, 58, 2429. Jensen, N. J.; Tomer, K. 6.; Gross, M. L. J. Am. Chem. Soc. 1985, 107. .- . , 1883. .- - -. Adams, J.; Gross, M. L. Anal. Chem. 1987, 5 9 , 1576. Jensen, N. J.; Tomer, K. 6.; Gross, M. L. L@& 1987. 2 2 , 480. Jensen, N. J.; Tomer, K. 6.; Gross, M. L. LIP& 1986, 2 1 , 580. p. A,; Crow, F. w.; Gross, M. L. Anal. che” 1985, 57, 2984. Tomer, K. 6.; Gross, M. L. Bbnmd. Environ. Mass. Spectrom. 1988, 15, 89.
Kenneth A. Caldwell Michael L. Gross* Midwest Center for Mass Spectrometry Department of Chemistry University of Nebraska-Lincoln Lincoln, Nebraska 68508
RECEIVED for review September 6,1988. Accepted December 2,1988. This research was supported by the National Science Foundation grant to the Midwest Center for M~~~ Spectrometry (Grant No. CHE-8620177).
Sorbent Isolation and Elution with an Immiscible Eluent in Flow Injection Analysis Sir: On-line preconcentration, e.g., of metal ions on an ion-exchange resin, followed by elution and detection has proved to be a versatile and sensitive technique for trace metal analysis, especially when coupled to atomic spectrometry (1-5). Various ion-exchange materials have been exploited for this purpose (5-9), and the utility of time-based, rather than valve-based, injection has been shown ( 2 , 3 , 5 ) . System configurations have been developed that prevent the sample matrix from entering the detector and allow elution to be carried out in a back-flushing mode (2, 7). The use of two preconcentration columns (one is in load mode while the other is in elution mode) has been introduced to increase sample throughput (2, 6, 7, 10). Although the advantages offered by such methods, namely, improvements in detectability and elimination of matrix effects, are desirable in many areas other than trace metal determinations, not many efforts have been made (11). While some ingenious membrane-based techniques for analyte isolation from the matrix and selective preconcentration have been developed (12-14), it is hard to equal the reliability of a packed-column-based device over extended periods of operation. Thus far, in all continuous analysis applications, the eluting solvents have been miscible with the sample matrix. The use of an immiscible solvent not only extends the scope of the general technique but also can provide unique selectivities and nearly ideal “plug elution”. In principle, the immiscible phase can be isolated with a membrane-based phase separator (see for example, ref 15 and citations therein). However, a potentially more robust approach is to actively sense the im0003-2700/89/0361-0496$01.50/0
miscible plug, isolate it, and accordingly direct it to the detector, using a miscible carrier and performing further chemistry en route, if desired. This paper demonstrates the above principle for preconcentration/sorption of mercaptans from a gasoline stream on an anion exchanger, elution by an aqueous alkali, and colorimetric determination in the aqueous eluate. A second example is shown for the preconcentration of aniline in a benzene stream on a cation exchanger, elution by dilute H2S04,and direct UV detection. EXPERIMENTAL SECTION Reagents. DTNB (5,5’-dithiobis(2-nitrobenzoicacid)) was obtained from Sigma Chemical Co. Various mercaptans were bought as neat liquids (Aldrich). All other reagents were of analytical reagent grade. Deionized distilled water was used throughout. “Regular”grade gasoline was purchased from local filling stations. Mercaptans were removed from gasoline by extracting with 5 M NaOH twice. This “clean”gasoline was used for establishing blank responses and as a medium for preparing mercaptan standards. Mercaptan standards in gasoline are not stable for more than 24 h. The standards were kept in ice during use.
Flow injection system configurationsinvolved a continuously flowing sample stream (rFIA, see ref 16);this arrangement is often advantageous for process applications. Experimental System. The mercaptan determination system is shown schematically in Figure 1. Gasoline, with or without added thiols, flows through a electropneumatically actuated rotary valve V1 @heodyne Type 5020, Cotati, CA) equipped with a WpL sample loop and a microcolumn (1.7 mm i.d. poly(tetrafluor0ethylene) (PTFE) tube, packed with a strong base anion ex0 1989 American Chemlcal Society
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Table I. Logic Output of V2 Delay Circuitry (Figure 3b)
temporal state initialized sensor activated sensor off tc ( 8 ) from previous step
heedle
R2
5
DTNB ImM 1
U XlOO
I
Waste
pL/min
Figure 1. Experimental arrangement for the measurement of mercaptans in gasoline. The inset shows the conductivity probes connected to V2. Pump
w/Aniline
Waste
Flgure 2. Experimental arrangement for the measurement of aniline in benzene.
changer, Amberlite IRA-40l-S,1650 mesh, Sigma Chemical Co.; bed height 5-15 mm, held in place by glass wool) and then through the load port of valve V2 to waste. Valve V2 is the same as V1 except for a 15-pL loop volume. The loop loading and waste ports of this valve contain stainless steel hypodermic needle inserts as shown in the inset of Figure 1. The conductivity between these two metallic probes is monitored; when the loop is filled with a conductive aqueous extract rather than the gasoline carrier, the electrical resistance drops and valve V2 is automatically switched to the inject position (vide infra). Typically, the system operates as follows. Gasoline flows at a fixed flow rate (330 pL/min) through the OH--form column, and mercaptans present in the gasoline are concentrated on the sorbent; meanwhile, the alkaline reagent, R1 (100 pL/min), fills the loop of VI. The totalvolume of sample preconcentrated before elution occurs is dependent on the inject/load cycling of V1. In this work, we used a 0.4/1.6-min inject/load cycle; the timing was provided by a home-built digital timer. Thus, the total volume of gasoline preconcentrated is the gasoline flow rate multiplied by the cycle time, less the loop volume of V1 (660-60 = 600 pL). When V1 is switched to the inject position, the alkaline slug R1 elutes the mercaptan captured on the microcolumn and regenerates the column. As this aqueous extract passes through valve V2, the aqueous phase is isolated by automatic actuation of the valve and put into a carrier stream of R1 (100 pL/min). A buffering reagent, R2, is then added at 500 pL/min and mixed in a knotted mixing coil (67 cm), and this procedure is followed by the addition of 1mM aqueous DTNB and a second mixing coil (45 cm). The optical detector (Model 757, Kratos, Inc., Ramsey, NJ; 8-mm path length, 6-nm bandwidth, 12-pL cell volume, heat exchanger removed to reduce back-pressure) is set at 412 nm. Since V2 is activated by the injected immiscible liquid from V1, the cycling period of V2 is the same as that of V1; i.e., a fresh extract is injected into the aqueous system every 2 min. The determination system for aniline in benzene is shown schematically in Figure 2. In strongly acid aqueous solutions, the best net signal due to the anilinium ion over a blank from water saturated with benzene is obtained by monitoring at 280
K1
U2a“
U3
U4b
open short open open
high high low low
low low high low
low high high low
‘U2a (output) = low = U2b = high = U2c = low = 555 (U3) trigger input, U3 triggers on falling edge. bU4 (outputs) = high s; injecK2 = short = V2 = inject. ‘U3 pulse period is 0.0011RSsn tion interval of V1 must be more than this chosen delay period. nm. Except for the blank response due to the dissolved benzene, no significant interference is encountered in typical benzene samples as the basic compounds are selectively preconcentrated on the acidic sorbent. This permits the simple direct detection approach reported here; obviously more specific aqueous chemistry,e.g., diazo coupling, can be carried out for situations requiring such specificity. Benzene, “clean” (twice extracted with 1 M H2S04) or containing a known concentration of aniline, was pumped pneumatically; the choice of clean or aniline-containing benzene was made by a three-way valve as shown. The benzene stream flows through valve V1 (loop volume 65 pL) and through a 20 X 1.5 mm bed of H+-form,strong acid type cation-exchange resin (Rexyn-lOl,50 mesh, Fisher Scientific) and then through the load port of valve V2 (loop volume 50 pL). Sulfuric acid, 0.1 M, acts as the eluting solvent and is injected through V1. The same reagent acts as the carrier to carry the aqueous segment, isolated by V2, to the detector. The valves operate on a 1/0.5min load/inject cycle. The detector, the same as that used in the mercaptan system, is operated at 280 nm. In both systems, all connecting tubing was 0.8 mm i.d. PTFE, and minimum possible lengths of connecting tubing were used for all lengths not explicitly specified. The pump shown in Figures 1 and 2 is a multichannel peristaltic pump (Model Minipuls 2, Gilson Medical Electronics, Middleton, WI). The automatic actuation of V2 is performed with the circuitry schematically shown in Figure 3. The status of filling of the V2 loop by a conductive liquid is monitored by the probes; the resistance across these probes constitutes one arm of a bridge circuit (Figure 3a). The bridge output is fed to the operational amplifier U1, used here as a voltage comparator. The variable 10-Ma resistor in the bridge provides a “sensitivity adjust” control and determines the probe resistance at which loop filling is sensed. As the probe resistance falls below this resistance, U1 turns on and causes driver transistor Q1 to activate the relay K1. A light-emitting diode is simultaneously turned on to indicate the sensor status. The normally open output terminals of K1 short as the sensor activates. This output is fed to the delay circuitry shown in Figure 3b. The delay circuitry is necessary for the following reason: A finite time is needed for the complete injection of the conductive slug isolated in the loop of V2. Even after V2 switches to inject, nonconducting organic liquid continues to flow and removes the conductive liquid located between each probe and the corresponding valve ports. With only the sensing circuitry described above, the valve will switch back to the load position, before the injection is completed. The circuitry in Figure 3b provides a user-selectable variable delay that maintains V2 in the inject position even after the sensor output is open; during this delay period, the sensor status is ignored. The truth table for the logic status is given in Table I. Although no explicit examples are presented in this paper, a similar arrangement has been successfully used for isolating a nonconducting liquid segment (e.g., an organic liquid) from a conducting carrier. In this case, both the probes are located immediately following the waste port of V2 and additional circuitry is required.
RESULTS AND DISCUSSION System Performance. The chemistry of the reaction for the determination of mercaptans involves the cleavage of the disulfide bond in DTNB by RS-, a nucleophile (I7). The reaction proceeds well under alkaline conditions; however,
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 5, MARCH 1, 1989
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-Tour 50 SCC.
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I
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Flgure 3. (a) Sensing circuitry for the status of loop filling by an immiscible segment. (b) Delay circuitry to maintain valve in inbct position after sensor goes off.
[OH-] becomes high enough at pH >11 to attack DTNB in a similar manner and undesirably increases the background absorbance. Our initial experiments indicated that effective elution and regeneration of the concentration column is attained only by using relatively high concentrations of NaOH as R1. This necessitated the use of 1 M NaHC03 as R2 as a buffering agent. With a 5 1 flow ratio for R2:R1, it was pmsible to maintain the final reaction pH in the range 8.5-10.5 as R1 was varied from 0.001 to 5.0 M NaOH. By direct injection of aqueous mercaptan standards through V2, it was established that the response of the system, either in terms of peak height or peak area, was essentially constant within this pH range (8.5-10.5) and showed only a small variation from one mercaptan to another. However, there was considerable influence of the NaOH concentration on the results for the overall isolation/elution system; the response of 1propanethiol (in mAU/mM) increased in the order 36.4 4, 103 0.7, 534 0.9, and 716 f 4 with 0.001,0.1, 1.0, and 5.0 M NaOH, respectively. This behavior is not due to a sharper frontal elution profile with more concentrated NaOH solutions; peak areas increase with increasing NaOH concentration as well. n-Alkyl mercaptans, C2-C5, were all found to show the same trend, albeit the absolute response differed from one
*
*
mercaptan to another, generally decreasing with increasing chain length. Typical system output is shown in Figure 4. As Figure 4 indicates, there is no indication of significantly greater peak tailing for the heavier mercaptans compared to that for the lighter ones; the changes in peak areas follow those of peak heights. Better response with increasing NaOH concentration may be due to better elution or better regeneration or both. However, even with the lowest concentration of NaOH used, the system provided enough sensitivity for the intended application and linear response to mercaptan concentrations in the C-2 mM range. The lack of equivalence of response from different mercaptans appears to be due to the differences in the capture efficiencies of the different mercaptans on the anion-exchangemicrocolumn. However, all C 1 4 5 mercaptans NaOH slug, appear to be quantitatively eluted by the 6 0 - ~ L at least with NaOH concentrations 1 1 M. Gasoline samples, collected from three separate filling stations (but of the same brand and grade) in the city, were measured and found to contain 0.52 0.02 mM mercaptan, on the basis of the response of the system to propanethiol. Some states have a maximum allowable concentration of sulfur in gasoline; for example, the State of California allows no more than 300 ppm sulfur (-9.4 mM) in unleaded gasoline (18).
*
Anal. Chem. 1989, 6 1 , 499-503
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termination system, subsequent chemistry needed to be performed. In the aniline determination system, the overwhelmingly large optical absorption from neat benzene and the tendency of one phase or the other to adhere to the detector window made it impossible to reliably monitor the absorbance of the aqueous segment without phase separation. However, there may be situations where immiscible solvent elution can be performed without the need to isolate the segment. For example, an aqueous sample stream can be merged with the stream of a suitable ligand solution, e.g., 8-hydroxyquinoline, and the resulting chelate taken up on a nonpolar sorbent. The sorbed metal complexes can be later eluted by a plug of an immiscible nonpolar solvent and conducted to the nebulizer of an atomic sepctrometer. The advantage, compated to that of miscible stripping reagents, is the lack of dispersion in the inevitable conduit between the preconcentration system and the atomic spectrometer.
LITERATURE CITED Flgure 4. System output for the mercaptan system. R1 is 1 M NaOH; R2 is 1 M NaHCO, (see Figure 1). A: blank. B, C, D, E: C5, C4, C3, and C2 n-alkyl mercaptans, 1 mM each.
The aniline determination system provided linear response in the range 0-10 ng/mL, with a detection limit (3 times blank noise over blank, as in ref 19) of 0.2 ng/mL and a calibration slope of 8.5 mAU/(ng/mL). Repeat injections of the benzene passed through the Catex resin showed that aniline was quantitatively taken up by the cation-exchange resin. The response tended to saturate above 10 ng/mL; it is likely that the nonpolar phase does not penetrate into the interior of the water-swollen resin beads, and thus the capacity for uptake is limited to surface sites. It is likely that a decrease in the particle size of the resin and/or an increase in the microcolumn dimensions can be used to extend the upper dynamic range. It is noteworthy, however, that the captured aniline is quantitatively eluted by the 6 5 - ~ Lslug of 0.1 M H2S04 with the present microcolumn. The process of zone sampling (20),wherein any desired portion of a dispersed sample is isolated by timed actuation of a valve, is well-known in flow injection analysis (15). In the present case, the desired zone is the immiscible liquid segment, and obviously a timed actuation of V2 can also be used. Active sensing of the segment to actuate V2 is, however, potentially advantageous. The immiscible plug containing the eluted analyte is axially inhomogeneous. In a time-based segment isolation scheme, any fluctuations in the flow rate will result in a slightly different cut of the segment being sampled by V2. Even with only 25% of the segment being sampled by V2, as in the mercaptan system, acceptable precision (typically 2-3% RSD) is obtained with the active sensing approach. On the other hand, for cases where sensitivity is a major issue, the majority of the liquid injected by V1 can be successfully isolated by V2. The difference between V1 and V2 loop volumes for the aniline system is only 15 p L and can conceivably be reduced further. Isolation of the immiscible segment was necessary in both of the cases presented in this paper. In the mercaptan de-
(1) Oisen, S.; Pessenda, L. C. R.; Ruzicka, J.: Hansen, E. H. Analyst 1983, 108, 905-913. (2) Hartenstein, S. D.; Ruzicka, J.; Christian, G. D. Anal. Chem. 1985, 57, 21-25. (3) . . Hartenstein. S. D.: Christian, G. D.: Ruzicka, J. Can. J. Spectrosc. 1986, 30,144-148. (4) Hirata, S.; Umezaki, Y.; Ikeda, M . Anal. Chem. 1988, 5 8 . 2602-261 1. (5) Maiamas. F.; Bengtsson, M.; Johansson. G. Anal. Chim. Acta 1984, 160. 1-10.
(6) Fang, Z.;Xu, S.; Zhang, S. Anal. Chim. Acta 1984, 164, 41-50. (7) Fang, 2.; Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1984, 164, 23-39. (8) Miiosavijevic, E. 8.; Ruzicka, J.; Hansen, E. H. Anal. Chlm. Acta 1985, 169, 321-324. (9) Bengtsson, M.; Maiamas, F.; Torstensson, A,; Regneii, 0.: Johansson, G. Mikrochlm. Acta 1985, I I I . 209-221. (10) Storgaard Jargensen, S.; Petersen, K. M.; Hansen, L. A. Anal. Chlm. Acta 1985, 169, 51-57. (11) Bergamin Filho, H.; Reis, B. F.; Jacintho, A. 0.; Zagatto,E. A. G. Anal. Chlm. Acta 1980, 117, 81-89. (12) Martin, G. €3.; Meyerhoff, M. E. Anal. Chim. Acta 1988, 186. 71-80. (13) Chang, Q.; Meyerhoff, M. E. Anal. Chim. Acta 1986, 186, 61-90. (14) Audunsson, G. Anal. Chem. 1986, 58, 2714-2723. (15) Ruzicka, J.; Hansen, E. H. Flow Inlection Analysis, 2nd ed.; Wiley: New York, 1988. (16) Johnson, K. S.; Petty, R. L. Anal. Chem. 1982, 5 4 , 1185-1187. (17) Ellman, 0.L. Arch. Blochem. Biophys. 1959, 82, 70-77. (18) Title 13, Section 2252, California Code of Regulations, State of California, Sacramento, CA. (19) American Chemical Society Committee on Environmental Improvement. Anal. Chem. 1980, 52. 2242-2249. (20) Reis, B. F.; Jacintho, A. 0.; Mortatti, J.; Krug, FJ.; Zagatto, E. A.O.; Bergamin Filho, H.; Pessenda, L. C. R. Anal. Chim. Acta 1981, 123, 22 1-228.
Wei Lei Purnendu K. Dasgupta* Jorge L. Lopez Department of Chemistry and Biochemistry Texas Tech University Lubbock, Texas 79409-1061
Don C. Olson Shell Development Company Houston, Texas 77251-1380 RECEIVED for review September 27,1988. Accepted November 30,1988. This work was supported by Shell Research and Development Co., Houston, TX.
Anion-Selective Electrodes Based on a Hydrophobic Vitamin B, Derivative Sic Vitamin BI2is a rather complex molecule that contains the essential trace element cobalt. In the isolated vitamin, also known as cyanocobalamin, the cobalt has a coordination
number of six. The four equatorial coordination sites are occupied by the nitrogens of the corrin ring, the axial ligands being a cyano group and the dimethylbenzimidazole ribo-
0003-2700/89/0361-0499$01.50/00 1989 American Chemical Society
