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As was indicated earlier, transient times exceeding 100 ms and resolution in excess of 100000 at m / z 1000 can easily be achieved if the pressure in the ion cyclotron resonance cell is kept below torr. In the present experiments the cell pressure could only be maintained a t 2 X lo-' torr, with or without sample in the ion source, because the system is still under construction and is now operating with temporary pumps of marginal capacity on the cell. In its final configuration the pumping system on the tandem-quadrupole Fourier transform instrument will maintain the cell pressure a t or below. Initial results presented in this and four earlier papers (16-19) provide us with considerable encouragement in our effort to develop a new approach to biopolymer sequencing using the tandem-quadrupole Fourier transform mass spectrometer. Previous work demonstrated that ions produced in a quadrupole ion source could be transmitted with high efficiency through the fringing fields of a superconducting magnet using rf only quadrupoles and then stored in an ion cyclotron resonance cell (17-19). Here we have shown that the tandem-quadrupole Fourier transform instrument can also be employed for mass analysis of ions produced by cesium ion bombardment of nonvolatile sample dissolved in a volatile liquid matrix. Laser photodissociation of peptide MH+ ions in an ion cyclotron resonance cell has recently been found to proceed with high efficiency and yield excellent sequence information (16). Thus it now seems reasonable to assume that large oligopeptides produced by either chemical or enzymatic cleavage of proteins can be completely sequenced a t the low picomole level by using a pulsed cesium ion gun to minimize sample consumption during the ionization process, rf only quadrupoles to transmit selectively only the high mass ions of interest, an ion cyclotron resonance cell operated at low pressure to accumulate and store MH+ ion produced in low abundance, laser photodissociation to generate fragment ions characteristic of the amino acid sequence, and Fourier transform mass spectrometry to produce a mass spectrum of the daughter ions a t high resolution. This same approach should also work well for the characterization of oligonucleotides, oligosaccharides, and glycoproteins.
ACKNOWLEDGMENT The authors are indebted to Jon Amy, Michael Story, and Robert Finnigan of Finnigan-MAT Corp. for their many contributions to the development of the tandem-quadrupole Fourier transform mass spectrometer. Registry No. Met-Arg-Phe-Ala, 67368-29-0;Leu-Trp-MetArg-Phe-Ala, 53214-98-5;angiotensin, 484-42-4; renin tetradecapeptide, 64315-16-8;cesium ion, 18459-37-5.
LITERATURE CITED (1) Hunt, D. F. Int. J . Mass Spectrom. Ion Phys. 1982, 45, 111-123. (2) Busch, K. L.; Cooks, R. G. Science 1982, 278,247-254. (3) Rinehart. K. L., Jr. Science 1982, 278,254-260. (4) Hunt, D. F.;Bone, W. M.; Shabanowitz, J.; Rhodes, G.; Ballard, J. M.
Anal Chem. 1981, 53, 1704-1706. (5) Fitton, J. E.; Hunt, D. F.; Marasco, J.; Shabanowitz, J.; Winston, S.; Dell, A. FEBS Lett. 1984, 769,25-29. (6) Comisarow, M. B.; Marshall, A. G. Chem. Phys. Lett. 1974, 25,
282-263. (7) McIver, Jr., R. T. A m . Lab. (Fairfield, Conn.) 1980, 12 (1l), 18-24. (8) Wilkins, C. L.; Gross, M. L. Anal. Chem. 1981, 53, 1661A-1676A. (9) Bowers, W. D.; Hunter, R. L.; McIver, R. T., Jr. Ind. Res.lDev. 1983, 25(11),124-128. (IO) Carlin, T. J.; Frieser, B. S. Anal. Chem. 1983, 55, 955-958. (11) McIver, R. T., Jr.; Bowers, W. D. I n "Tandem Mass Spectrometry"; McLafferty, F. W., Ed.; Wiley: New York. 1983;pp 287-301. (12) Cody, R. B.; Burnier, R. C.; Cassady, C. J.; Freiser, 8. S. Anal. Chem. 1982, 54, 2225-2228. (13) Dunbar, R. C. I n "Gas Phase Ion Chemistry"; Bowers, M. T., Ed.; Academic Press: New York, 1979;pp 181-220. (14) Thorne, L. R.; Wright, C. A.; Beauchamp, J. L. I n "Ion Cyclotron Resonance 11"; Hartmann, H., Wanczek, K. P., Eds.; Springer-Verlag: Berlin, 1982;pp 43-97. 15) Jasinski, J. M.; Rosenfeld, R. N.;Meyer, F. K.; Brauman, J. L. J . A m . Chem. SOC. 1982, 704, 652-658. Delbert, S. S.;Hunter, R. L.; McIver. R. T.. Jr. J . A m . 16) Bowers, W. 17.; Chem. SOC.,in press.
17) McIver, Jr., R. T.; Hunter, R. L.; Story, M. S.;Syka, J.; Labunsky, M.; Presented at the 31st Annual Conference on Mass Spectrometry and Allied Topics, Boston, MA, 1983. 18) McIver, R. T., Jr.; Hunter, R. L.; Bowers, W. D. Presented at the "32nd Annual Conference on Mass Spectrometry and Allied Topics", San Antonio, TX, 1984. (19) McIver, R. T., Jr.; Hunter, R. L.; Bowers, W. D. Int. J , Mass Spectrom. Ion Phys ., in press, (20) Abreth, W.: Straub, K. M.; Burlingame, A. L. Anal. Chem. 1982, 54,
2029-2034.
Donald F. Hunt* Jeffrey Shabanowitz Department of Chemistry University of Virginia Charlottesville, Virginia 22901
Robert T. McIver, Jr.* Richard L. Hunter Department of Chemistry University of California Irvine, California 92717
John E. P. Syka Finnigan-MAT Corp. 355 River Oaks Parkway San Jose, California 95134 RECEIVED for review September 4,1984. Accepted November 28, 1984. This research was supported by grants awarded to D.F.H. from the NIH (AM26533 and a Fogarty Senior International Fellowship) and to R.T.M. from the National Science Foundation and by a gift to D.F.H. from the Monsanto co.
Hollow Fiber Postcolumn Reactor for Liquid Chromatography Sir: A great deal of attention has been focused on postcolumn derivatization for enhanced detection of species eluting from liquid chromatography columns. Frei and Scholten ( I ) have reviewed many of the theoretical and technical aspects of reaction detectors, including the problems associated with their use, while Takata and Muto ( 2 ) first discussed reaction detectors for uses such as enhanced electrochemical detection. Hollow fiber membranes have been reported for postcolumn reaction in both unpacked and packed hollow fiber suppressors for ion chromatography (3, 4).
The requirements for good postcolumn derivatization can be reduced to three key components: (a) reagent introduction, (b) delay or reaction/mixing loops, and (c) proper detection devices. Both (b) and (c) are essentially fixed by the reaction time/temperature and chemistry chosen. Part a, the reactor itself, allows more flexibility. For example, with reagent mixing, techniques such as air segmentation, pumping reagent into a mixing device, and packed beds of reagent granules have all been used. Unfortunately, each of these methods has its drawbacks. Air segmentation and mixing devices require an additional pump and lead to band spreading and dilution,
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reactor. reducing the sensitivity gained through derivatization. Packed beds of reagent require a sparingly soluble reagent, usually deplete rapidly, and also cause band spreading, particularly as the reagent depletes. We have developed new reactor devices which eliminate or control the problems associated with other reactor designs. These devices are easily adapted to conventional liquid chromatography equipment and are applicable to a wide variety of reagent and reactor systems (5, 6). EXPERIMENTAL SECTION Apparatus. With the exception of the hollow fiber devices, commercially available liquid chromatography equipment was used in all cases. Chromatographic conditions will be presented in the Results and Discussion section for each system. A typical hollow fiber reactor, shown in Figure 1,was composed of a loop of five, 325 fim i.d. sulfonated polyethylene hollow fibers 8 in. long. After being attached to suitable connecting fittings, this loop was suspended in a container of the appropriate reagent. Typical ion exchange capacity was 1mequiv/g on a dry weight basis. Eluent flow was through the fibers. Means for preparing the fibers and devices are found elsewhere (3, 4). For experiments with fluorescarnine (4-phenylspiro[furan-2-(3H),1’(3’H)-phthalan]3,3’-dione), hollow fibers of 60% poly(dimethylsilane), 40% amethylstyrene copolymer were used (SR-a/751-7010from Bio-Rad Laboratories). Reagents. HPLC grade solvents and 0.45 Hm filtered water were used for reagent solution and mobile phase preparation. Chemicals used were ACS reagent grade or better and were obtained from commercial sources (except for samples of the nitrophenols which were obtained from Dow Chemical USA, Midland, MI).
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Flgure 2. Comparison of nitrophenols with (A) 280 nm detection, no postcolumn reactor, and eluent pH of 6.0; (B) 410 nm detection, 0.1 N NaOH pumped into tee postcolumn, and pH of 10 at detection: and
(C) 410 nm detection, hollow fiber reactor, and pH 10 at detection.
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Flgure 3. Comparison of band spreading with (B) and without (A) the hollow fiber reactor.
Figure 2 illustrates the results obtained for three experiments: (A) detection at 280 nm with no pH change; (B) detection at 410 nm with 0.1 N NaOH pumped into a mixing tee at 10% of the eluent flow rate; and (C)detection at 410 nm using the holow fiber reactor to adjust the eluent to pH 10. Chromatographic conditions were as follows: column, Partisil SAX 10/25; eluent, 12% methanol/water with 0.08 M sodium perchlorate and 0.104 M ammonium acetate adR E S U L T S AND DISCUSSION justed to pH 6.0 with glacial acetic acid flow rate, 2.0 mL/min; Enhanced Detection of Nitrophenols. Rarely is the best injection size, 100 kL. The sample used in these experiments was a synthetic aqueous waste stream with high levels of pH for a particular separation also the optimum for detection. organics spiked to 1ppm with each of the nitrophenols. Note A case in point is that of substituted nitrophenols such as 2,4-dinitro-6-sec-butylphenol (2,4-DNP) and its related sulin particular the improved base line stability and enhanced fonated compounds 3-nitro-4-hydroxy-5-sec-butylbenzene- sensitivity to 4-OH when the hollow fiber reactor is used. sulfonic acid (4-OH) and 2-hydroxy-3-nitro-5-sec-butylThe chromatograms in Figure 3 illustrate the excellent flow benzenesulfonic acid (2-OH). An aqueous solution containing characteristics of the hollow fiber reactor. The 2,4-dinitrotrace amounts of these three compounds and large amounts 6-sec-butylphenol chromatography is shown with and without of other UV absorbing organics can be separated by conventhe hollow fiber reactor in line. The detection wavelength was tional HPLC on a Partisil ODs-2 column using a mobile phase 280 nm and no pH change was performed so that the peak adjusted to pH 7.0. However, a large tailing peak eluting with area would remain unchanged and only the peak broadening the sample solvent front reduces the detection limit when 280 due to fiber flow dynamics would be measured. The calculated nm is used as the detection wavelength. By inserting the number of theoretical plates was found to be 2000 in the hollow fiber postcolumn reactor shown in Figure 1between absence of the hollow fibers (A) and 1940 with the hollow the column and the detector and filling the reactor with 2 N fibers in line (B) indicating less than a 5% loss of resolution ammonium hydroxide, resulting in permeation of ammonia due to the reactor. Chromatographic conditions were given through the fiber wall into the eluent, the pH of the eluent in Figure 2. Applications of this device in the determination is raised to 10 and 410-nm detection of the ionized phenols of nitrophenols in wastewater allowed detection in the 25 to is possible. This reduces the interferences from other organics 100 ppb level with excellent linearity over 2 orders of magseen at 280 nm and resuls in enhanced detection of the ninitude and precision of 6% or better (95% confidence level) trophenols. a t the 1 ppm level with a fixed wavelength detector.
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Enhanced Fluorescent Detection of Phenols. The fluorescence of some phenols is known to be pH dependent (71, usually increasing in intensity with increasing pH a t a given pair of excitation/detection wavelengths. Unfortunately most HPLC columns are limited to a maximum pH of about 8 due to their silica base material. A hollow fiber reactor similar to that in Figure 1was used to increase the pH of the eluent from a phenol separation. Figure 4 shows the effect of changing the eluent pH from 2.0 (A) to 10.0 (B) on the detection of a mixture of hydroxycarboxybenzenes. Fluorescence excitation was a t 320 nm with detection at 440 nm. Chromatographic conditions were as follows: eluent, gradient starting a t 0.2 M phosphoric acid in water and proceeding to 0.2 M phosphoric acid in 20% acetonitrile/ water; injection size, 10 pL; flow rate, 1.0 mL/min; column, Zorbax ODs. Note the selective increase in the sensitivity for some compounds without a concomitant loss of resolution. Fluorescent Detection of Amines. Detection of primary amines by reaction with fluorescamine to form fluorescent products is a well-established technique (8). This system was chosen to show the efficiency of the hollow fiber postcolumn reactor when used in a chemical reaction mode. The reaction proceeds rapidly with 2 mg/mL of fluorescamine in acetonitrile solution outside the membrane. Figure 5 is a comparison of two 10-pL injections of 1mg/mL benzylamine onto a Waters micro-C18 column. The mobile phase was 30% acetonitrile/water 0.01 M in ammonium acetate with a flow rate of 2.0 mL/min. The letter A denotes the point at which an injection was made prior to the addition of reagent to the fiber system while letter B is the point at which reagent was added to the reactor. A second injection of amine was made a t point C. The rapid reaction of fluorescamine and primary amines makes this application ideally suited for postcolumn reaction since a delay or reaction coil is not required. Also of interest is the rapid equilibration of the hollow fiber unit upon addition of the reagent. No major base line disruption is observed and base line noise is not significantly increased. Reaction of Ninhydrin with Amino Acids. A much slower reaction, ninhydrin color formation with amino acids, was also studied and satisfactory performance achieved with the hollow fiber postcolumn reactor. A 90 OC delay/reaction coil was used prior to the detector. The delay coil provided a 3-min delay a t a flow rate of 0.52 mL/min and was approximately 2 m in length. Two-hundred microliter injections of 50 ppm glycine were made to simulate the elution of amino acids from a chromatography column. The eluent was 0.02 M citric acid in water adjusted to pH 4.0 with NaOH. The device illustrated in Figure 1was used as the reactor with 10% ninhydrin in water on the outside of the fibers. Only 75%
Time in Minutes
Figure 5. Effect of fluorescamine addition on detection of benzylamine.
reaction of the amino acid with ninhydrin was observed under these conditions; however, with the use of either internal standards or with alternating injections of standards, the excellent reproducibility and stability of this system allowed the detection of amino acid with good precision. Detection limit of glycine using this system is in the parts-per-million range and illustrates the use of the hollow fiber reactor in a case where complete reaction is not necessary for useful detection enhancement. (Simulated chromatograms are not shown.) Selection and Use of Hollow Fibers. There are several considerations in making and applying a hollow fiber reactor to a particular system. The type of membrane material should be chosen for maximum reagent flux with minimum sample loss and be compatible with the solvent and mobile phase used. Since, however, most peaks eluting from chromatography columns are relatively dilute, it is possible to use membranes with virtually no permeation selectivity of the reagent over that of the sample material. This is principally due to the first-order nature of membrane permeation which results in roughly similar percentages of material permeating when in contact with the membrane (assuming approximately the same molecular weights). This allows the use of high concentrations of reagent on the outside of the membrane to permeate only a small percentage through the membrane but resulting in a high concentration in the eluent. Correspondingly, only a small percentage of the sample permeates into the reagent chamber during its passage through the fibers. A primary consideration and problem in preparing these units is connection of the fibers into the LC system. Due to the wide variety of fittings available, there is no universally acceptable configuration. Units used in this laboratory have generally had the fibers potted into a fitting with a 1/4-28 male thread, using an appropriate adapter to 1/16 in. narrow bore tubing. Potting materials of choice have been silicone rubber, fluorosilicone rubber, and epoxy-selected depending upon the solvent resistance required. In multiple fiber units, to minimize band spreading, there must be equal flow through all fibers of the unit. This requires the fibers to be the same length, uniform in inner diameter, and have no flaws which restrict flow. To promote equal flow, it is desirable to keep a minimum pressure drop of 2-5 psi across the unit; this can be controlled by the number of fibers, length of the fibers, and fiber size. Note that these parameters will also affect the residence time of the sample in the unit
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and also the flux of the reagent into the eluent flow stream. Band spreading due to unequal fiber lengths or diameters in multifiber units can be minimized by adjusting the size of the hollow fiber unit so that i t has a total volume inside the fibers which is smaller than the volume of the peaks eluted. In this case only moderate end effects on the sample peaks will be observed in most systems. Reagent Addition. Several parameters should be considered for reagents in postcolumn reactions. The flux rate through a membrane is dependent upon the concentration gradient across the membrane. Thus as the reagent is depleted from the reservoir, the flux into the eluent will decrease. The effect this has on a particular analysis will depend on the system being studied. For example, if a large reservoir is used or a high reagent concentration is used to permeate excess reagent into the eluent, little effect will be seen on the analysis. Clearly the point should be determined for a given system where reagent depletion begins to affect the analysis and regeneration of the reagent performed prior to that time. Alternatively, a slow flow of fresh reagent may be used to replenish the reservoir by flowing around the fiber bundle. Attention should also be given to the choice of solvent used for the reagent as it may have either a benificial or detrimental effect in the flux of reagent through the membrane. Other Potential Uses for Hollow Fiber Reactors. This new technique of passive reagent addition or p H change is also applicable to electrochemical, turbidimetric, and conductometric detection as will as to vibrational and electronic
wavelength detection. These devices may be used both following chromatography and in flow injection analysis applications. The use of two or more reactors and multiple detectors may be desirable with particularly difficult sample matrices. Registry No. 2,4-DNP, 88-85-7; 4-OH, 93037-41-3;2-OH, 94042-50-9;benzylamine, 100-46-9;glycine, 56-40-6.
LITERATURE CITED Frei, R. W.; Scholten, A. H. M. T. J. Chromatogr. Sci. 1979, 17, 152-160. Takata, Y.; Muto, G. Anal. Chem. 1973, 4 5 , 1864-1868. Stevens, T. S.;Davis, J. C.; Small, H. Anal. Chem. 1981, 53, 1488- 1492. Stevens, T. S.; Jewett, G. L.; Bredeweg, R. A. Anal. Chem. 1982, 54, 1206-1208. Davis, J. C. United States Patent 4448691, 1984. Peterson, D. P.; Davls, J. C. United States Patent 4451 374, 1984. Schulman, S. G. Rev. Anal. Chem. 1971, 1 , 85-111. Udenfriend, S.;Stein, S.; Bohlen, S.; Dairman, P.; Leimgurber, W.; Weigele, M. Science 1972, 178, 871-872.
’
Present address: Surface Sclence Laboratories, Standard Oil (Ohio), Cleveland, OH 44128. ‘Present address: PPG Research Center, Barberton, OH 44203.
James C. Davis*’ Dennis P. Peterson2 Dow Chemical USA Midland, Michigan 48640 RECEIVED for review January 27, 1983. Resubmitted October 15, 1984. Accepted November 15, 1984.
Adding Forward Searching Capabilities to a Reverse Search Algorithm‘ for Unknown Mass Spectra Sir: Mass spectrometry, especially with direct coupling to the gas chromatograph (GC/MS), is the most widely used tool for the identification of unknown organic molecules. The modern GC/MS can produce dozens or hundreds of unknown electron-ionization mass spectra per hour, for which many computer matching systems have been proposed (1-13). Of these, it is well-known that forward searching algorithms are substantially superior for unknown mass spectra of pure compounds (1-3) and that reverse searching (4-6) is advantageous for spectra of mixtures. We describe here a combination of forward and reverse searching that shows substantially improved performance for spectra of both pure compounds and mixtures without prior knowledge of purity. Reverse searching, which is considered necessary even for capillary column GC/MS ( I , 2,14), gives matching credit only for peaks of the reference spectrum that are in the unknown, not vice versa. Because of this, matching an unknown spectrum of pure dioctyl phthalate retrieves those of octenes (Table I), a logical impurity, as the fragmentation of C8HI6 in the phthalate is the source of many peaks in its spectrum similar to those from octenes (Figure 1). This causes the reliability value (the probability that the degree of match found indicates the correct answer) predicted by the probability based matching (PBM) system (4, 6,8,12, 13,15-17) to be misleadingly high (32%) for 3-octene; 14 of the top 20 matches are C,H2,, Compounds. However, these would be rated poor matches by forward searching, which would also require the presence of phthalate peaks. The scheme described here for combining reverse and forward searching capabilities is based on the automatic subtraction of matching
reference spectra (8).
EXPERIMENTAL SECTION Calculations were carried out with a Hewlett-PackardHP-1000 computer, matching against a library of 76 663 different mass spectra of 67 128 different compounds from the Wiley/NBS data base (13, 18) (spectra of isotopically labeled compounds were omitted). A total of 392 different compounds, each of which was represented in the file by more than one different spectrum, were selected at random from the file. The spectrum of highest “quality index” (19) of each compound was used to make the “pure spectrum” file of unknowns. Groups of three of these spectra, ordered by increasing molecular weight, were combined in 60:3010 (totalions) proportion for the fde of 130 unknown mixture spectra. Reference spectra used as unknowns were omitted from the PBM results. The PBM program used (“system a”) includes several modifications of the original ( 4 , 6 ) ,such as ranking of retrieved spectra by reliability values (15),abundance based flagging of spurious peaks (15-1 7), and quadratic scaling to minimize experimental abundance variations (16, 17). Class I matches are the same compound or a stereoisomer; class IV matches represent compounds that are sufficiently similar structurally to make difficult their differentiation by mass spectrometry (6,15).For retrieved spectra the maximum “% contamination” (% C) and minimum “% component” values allowed are 80% (75% for Table I) and l o % , respectively, restricting the retrieval of minor mixture components. In forward searching modification “b” the predicted reliability value is adjusted t o reflect the magnitude of the %C value; the adjustment is based on PBM results using the 392 unknowns (17). For the final system “c”,each reference spectrum which matches with a predicted reliability value >lo% is subtracted from the 0 1985 American Chemical Soclety
