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Anal. Chem. 1991. 63. 296-299
after hydrolysis. Recovery percentages for polyamines in spiked serum were as follows: PUT, 100%; CAD, 93%; SPD, 104%; SPM, 94%. These data indicate that a sufficient excess of reagent was added to serum such that the yield with polyamines was not affected.
CONCLUSIONS It has been shown that 2-(9-anthryl)ethyl chloroformate (AEOC) is a sensitive and convenient precolumn derivatizing reagent for the determination of polyamines by HPLC and fluorescence detection. AEOC-derivatized polyamines are very stable, and the detection limits are more favorable than those reported previously for polyamines derivatized with FMOC. This finding indicates that the absorption spectral differences between the anthracene and fluorene chromophores allow for lower detection limits of AEOC-derivatized polyamines. Application of the derivatization procedure to the analysis of physiological fluids, therefore, represents an attractive method for biomedical studies.
ACKNOWLEDGMENT We thank Milissa A. Bolcar for providing valuable supporting chromatographic data. Registry No. AEOC, 129948-83-0;putrescine, 110-60-1;cadaverine, 462-94-2; spermidine, 124-20-9;spermine, 71-44-3.
LITERATURE CITED (1) Veening, H.; Pitt, W. W., Jr.; Jones, G., Jr. J . Chromafogr. 1974, 90, 129. (2) Sayem-eCDaher, N.; Simard. R. E.: L'Heureux, L.; Roberge, A. G. J . Chromatogr. 1983, 256,313. (3) Tabor, H.. Tabor, C. W., Eds. Po/yamines; Methods of Enzymology; Academic Press: New York, 1983, Vol. 94, pp 1-48. (4) Slmpson, R. C.; Mohammed, H. Y.; Veening, H. J . Liq. Chromatogr. 1982, 5 , 245. (5) Heideman, R. L.; Fickling, K. 5.; Walker, L. J. Clin. Chem. 1984, 30, 1243. (6) Price, J. R.; Metz, P. A,; Veening, H. Chromatographia 1987, 2 4 , 795.
(7) Morler-Teissler, E.; Drieu, K.; Rips, R. J . Li9. Chromatogr. 1988, 1 7 , 1627. (8) Kamei, S.; Ohkubo, A,; Saito, S.; Takagi, S. Anal. Chem. 1989, 6 1 , 1921. (9) Einarsson, S.; Josefsson, E.; Lagerkvist, S. J . Chromatogr. 1983, 282, 609. (10) Einarsson, S.; Folestad, S.: Josefsson, E.; Lagerkvist. S. Anal. Chem. 1986, 58, 1638. (11) Einarsson, S.; Josefsson, B.; Moiler, P.; Sanchez, D. Anal. Chem. 1987. 59. 1191. (12) Creech,-H. J.-Norman, R. N. J . Am. Chem. SOC. 1941, 6 3 , 1661. (13) Sango. C.; Zimerson, E. J . Liq. Chromafogr. 1980, 3,971. (14) Yoshida, T.; Uetake, A.; Murayama, H.; Nimura, N.; Kinoshita, T. J . Chromatogr. 1985, 348, 425. 15) Korte, W. D. J . Chromatogr. 1982, 243, 153. 16) (a) Goto. J.; Saito, M.; Chikai, T.; Goto, N.; Nambara, T. J . chfOm8togr. 1983, 276, 289. (b) Goto, J.; Goto, N.; Shamsa, F.; Saito, M.; Komatsu, S.; Suraki, K.; Nambara, T. Anal. Chim. Acta 1983, 147, 397. 17) (a) Langeman, H.; Hulshoff, A.; Underberg, W. J. M.; Offerman, F. 8 . J. M. J . Chromafogr. 1984, 290,215. (b) Baty, J. D.; Pazouki. S.;Dolphin, S. J . Chromatogr. 1987, 395,403. 18) Roach, M. C.; Ungar, L. W.; Zare, R. N.; Reimer. L. M.; Pompliano, D. I.; Frost, J. W. Anal. Chem. 1987, 59, 1056. (19) (a) Bayliss, M. A. J.; Homer, R. 8.; Shepherd, M. J. J . Chromafogr. 1988, 445, 393. (b) Bayliss, M. A. J.; Homer, R. B.; Shepherd, M. J. J . Chromatog. 1988, 445, 403. (20) Goto, J.; Ito, M.: Katsuki, S.; Saito, N.; Nambara, T. J . Li9. Chromatogr. 1986, 9 ,683. (21) Kornblum, N.; Scott, A. J . Org. Chem. 1977, 42, 399. (22) 'Sorensen, H. Ph.D. Thesis, University of Gothenburg (Sweden), 1989. (23) (a) Mikhailov, E. M. Izv. Akad. Nauk SSSR Ser. Khlm. 1948, 420423; Chem. Abstr. 1949, 43, 208g. (b) Becker, H.-D.; Hansen, L.; Anderson, K. J . Org. Chem. 1986, 51, 2956. (24) Calvert. J. G.; Pitts, J. N., Jr.; Phofochemistry; John Wlley 8 Sons, Inc.: New York, 1966; p 310. (25) Josefsson. B., et ai. Private communication, 1990 (manuscript In p r e p aration). (26) Moller, P. Eka Nobel, Nobel Industries. S-44501 Surte, Sweden, private communication, 1989. (27) Dorschel, C. A.; Ekmanis, J. L.; Oberholtzer, J. E.;Warren, F. V., Jr.; Bidlingmeyer. E. A. Anal. Chem. 1989, 6 1 , 951A.
RECEIVED for review August 6, 1990. Accepted November 8, 1990. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of this research.
CORRESPONDENCE Separation of Particles in Nonaqueous Suspensions by Thermal-Electrical Field-Flow Fractionation Sir: The separation and characterization of diverse categories of particles (ranging from to IO2pm in diameter) suspended in aqueous media by sedimentation field-flow fractionation (SdFFF) (1-9) and flow field-flow fractionation (FIFFF)(10-14) have been widely reported. By contrast, the pressing need to develop techniques for the characterization of particles suspended in organic liquids (including various oils) has received scant attention in FFF research, despite the fact that the FFF process is intrinsically as effective in organic as in aqueous suspensions. The few applications of SdFFF reported-using such liquid carriers as ethanol (15) and binary mixtures of ethanol and 1,1,2-trichlorotrifluoroethane(16)have failed to catalyze rapid developments in this area, in part because of concerns about seal degradation and solvent leakage. Concurrent with the development of SdFFF, thermal field-flow fractionation (ThFFF) evolved as a powerful technique for the separation and analysis of lipophilic polymers
dissolved in various organic solvents (3,17-19). Many linear and branched polymers and copolymers have been fractionated by this subtechnique of FFF, some of them with molecular weights ranging up to 50 X lo6 and higher (20). However, the separation of particles by ThFFF has not previously been achieved. (In this paper particles are distinguished from polymers by their rigid or semirigid three-dimensional structures.) We report here the first successful application of thermal FFF to the separation of particles suspended in nonaqueous (as well as aqueous) liquids. We furthermore report that the separation of particles can be achieved (or augmented) by applying a few volts of electrical potential across the channel, which acts in place of (or in addition to) applied temperature gradient in a conventional thermal FFF apparatus. Electrical FFF (ELFFF) has not previously been observed in nonaqueous media. The electrical component of retention provides another degree of freedom for broadening the basis of selectivity.
0003-2700/91/0363-0296$02.50/00 1991 American Chemical Society
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EXPERIMENTAL SECTION Like previously reported thermal FFF systems, the present apparatus uses a thin (76 pm) channel sandwiched between chrome-plated copper bars (21). The channel length (tip to tip) is 44.6 cm, and the breadth is 2.0 cm; the void volume is 0.71 mL. The upper bar was heated by means of inserted heating rods while the lower bar was cooled by the circulation of tap water through a network of cooling channels. The lower block was electrically grounded by virtue of its connection to the copper tubing supplying the tap water. A model 240A dc power supply from Keithley Instruments (Cleveland,OH) was used to apply a voltage between the two bars. The carrier solvents were delivered by a Waters Associates (Milford, MA) Model M-6000A pump. A 254-nm UV detector, Model UV-106A1, from Cole Scientific (Calasabas,CA) was used to detect eluting particles. The detector signal was fed to a Houston Instruments (Austin, TX) chart recorder. A pressure regulator was used to hold the channel outlet 100 psi above atmospheric pressure; this provides solvent stability when the operating temperature is near or above the solvent boiling point. Three reagent grade nonaqueous solvents were used in this investigation: tetrahydrofuran (THF),acetonitrile, and methanol. These solvents were obtained from EM Industries (Cherry Hill, NJ). Doubly distilled water was also used. All these solvents permit the transmission of UV light through the detector cell at 254 nm. A variety of particles were used in this work. Silica particles of diameters 0.05,0.10,0.15,0.25, and 0.5 pm were provided by J. N. Kinkel of E. Merck, FRG. Several sizes of polybutadiene latex particles were given us by Grant Von Wald of Dow Chemical Co. Polystyrene microspheres ranging in diameter from 0.091 to 7 pm were obtained from Duke Scientific. Polyvinyl toluene (0.399 pm) and PMMA (0.586 pm) latex particles were obtained from Seradyn while several silver halide colloidal samples were gifts of D. E. DeCann from Eastman Kodak. Aluminum silicate colloids in the size range 0.2-3.0 pm were obtained from Duke Scientific.
THEORY FFF is effective only if the field or gradient acting at right angles to flow generates sufficient force to drive different particle populations into thin transverse distributions that differ enough from one another in position to be differentially displaced by flow. For submicron particles and polymers, generally subject to the normal operating mode of FFF, the retention time t , of a particle is related to the force F acting on it by (3, 22)
t, = t o / E ( c o t h
2kT
Fw
where t o is the emergence time of a nonretained marker, k T is thermal energy, and w is the channel thickness. Commonly, Fw >> k T , in which case t , becomes linear in F according to the simple form t, = Fwto/6kT
(2)
The effective force FT generated by a temperature drop A T applied across the FFF channel is approximated by (3)
FT = fDTAT/W
(3)
where DT is the thermal diffusion coefficient and f is the friction coefficient. I f f is expressed in terms of particle diameter d and viscosity t by Stoke's law, f = 3 a t d , then eqs 2 and 3 yield the approximate retention time
t, = x t o ~ d D ~ A T / 2 = k TCTAT
NO.3, FEBRUARY 1, 1991 297
(4)
If the particle has an effective charge q and is subjected to a uniform electrical field E = V J w , then an additional force will act of magnitude FE = qVc/W = qI/KbL (5) where in the last expression FE is given in terms of current
I
0
20
40
60
80
TIME (mid
Figure 1. Separation of submicron size silica particles (of indicated diameters) suspended in acetonitrile by thermal FFF at AT = 52 K.
Z and specific conductivity K instead of channel voltage V,, which will differ from the applied (measured) voltage due to the potential drop through the double layers at each wall. The channel breadth and length are b and L , respectively. The net force on a particle will now be F = FT + FE, and t, will be given by eq 1 or 2. Using eq 2, t , is approximated by where constant CE = t o q w / 6 k T ~ b L .
RESULTS AND DISCUSSION Our initial studies were carried out by using only a thermal field. All of the particle types listed above were retained using acetonitrile as the carrier with A T values from 7 to 106 K. The inorganic particles were also retained in T H F but less so than in acetonitrile. The latex particles could not be tested in T H F because of their solubility in that solvent. The larger (>l-pm diameter) polystyrene latex particles were retained in water and methanol although the submicron polystyrene latex was not observably retained in these two solvents. Overall, acetonitrile proved to be the best of the four solvents for retention and separation. In order to confirm that the polystyrene latex particles did not swell in acetonitrile, the larger latex (d > 1hm) was boiled in acetonitrile for 2 h. Optical microscopy showed that there were no significant dimensional changes in the latex particles. Figure 1 illustrates the separation of silica particles of 0.05and 0.25-pm diameters suspended in acetonitrile by thermal FFF operating at A T = 52 K and at a flow rate V of 0.2 mL/min. The resolution, while satisfactory, would likely be better for narrower distributions of silica particles. Figure 2 shows the separation of four different sizes of polystyrene latex particles by thermal FFF a t a A T of only 17 K. In this case the flow rate of the acetonitrile carrier was 0.3 mL/min. Figure 3 shows the effect of a small electrical field on retention in acetonitrile. The three-peak profiles in this figure show the detector response for 0.198-pm latex particles eluting from the channel at A T = 17 K and V = 0.6 mL/min. The central peak (unbroken line) shows the retention obtained with no electrical field applied. The left-hand profile (broken line) shows the resulting peak with the application of 2 V (hot wall positive relative to cold wall) across the channel. The dotted peak profile on the right illustrates the enhanced retention achieved with -2 V applied. The electrical field clearly has a substantial effect on retention. The shifts in retention correspond in direction to those expected for negatively charged particles. Figure 4 shows that a small potential drop (-2 V) is by itself capable of inducing retention and separation. In this figure,
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
0.43u m
-.-
0.28pm
40
c
E
0.20pm 0.1ipm W W
1
0
20
io
30
40
0
TIME (mid
Flaure 2. SeDaration of four sizes of polystyrene latex microspheres suspended in’ acetonitrile by thermal FFF & AT = 17 K.
20
IO
30
50
40
60
AT (K)
Flgure 5. Plot of retention time t , against AT for three sizes of polystyrene latex beads suspended in acetonitrile.
30
Y
L
io
0
5
IO
15
TIME (mid
Figure 3. Influence of small electrical potentials (expressed as the voltage applied to the hot wall relative to the cold wall) on the elution profile of 0.198-pm polystyrene latex beads suspended in acetonitrile at AT = 17 K.
I
0.09pm
TIME (min)
Flgure 4. Electrical FFF separation of two sizes of polystyrene microspheres suspended in acetonitrile with -2-V electrical potential applied across the thermal FFF bars (AT = 0). polystyrene latex beads of diameters 0.091.and 0.426 pm are separated in acetonitrile with A T = 0 and V = 0.35 mL/min. Several plots were made to test the applicability of the theoretical equations. In Figure 5, retention time t, is plotted against AT for polystyrene latex beads of diameters 0.426, 0.198, and 0.091 pm. These measurements. (one per plotted point) were carried out in acetonitrile a t V = 0.3 mL/min. The plots verify a linear relationship between t , and A T , as expressed by eq 4. However, the intercepts are not zero. A more exacting theory based on eq 1 (rather than eq 2) shows that the intercept should be slightly positive, namely at t0/3, in this case 0.8 min (23). The actual intercepts are observed to be larger than this, particularly for the larger diameters. Finally, in Figure 6 t r is plotted against current I for the same three latex particles at a constant A T of 17 K. The solvent is again acetonitrile at V = 0.3 mL/min. The lines exhibit a roughly linear relationship, in this case in agreement
-2
-i
,
I 0
I
1
2
I (ma) Figure 6. Plot of retention time t , against current I for three sizes of polystyrene latex microspheres suspended in acetoniMle and subjected to a temperature drop of AT = 17 K.
with eq 6. (The three voltages used in these experiments are -2,0, and +2 V.) While the results given here appear to align themselves with the theory of normal mode operation in FFF, a number of important questions remain unanswered. First of all, neither of the coefficients in eq 6 are accessible to theory because neither the thermal diffusion properties (i.e., DT)of particles that control C T nor the electrical properties (i.e., q ) of the particles that control CE are well understood. Because these coefficients are not known, it is not clear how t, depends upon particle size and particle and carrier composition. Once these dependencies are better understood, it is likely that the thermal and electrical fields could be used in varying proportions to maximize not only size selectivity but also selectivity based on compositional differences of particles. The electrochemistry associated with the electrical component of FFF must also be better characterized and controlled. Some observed degradation of the channel walls might be alleviated by introducing into the solvent one or more components that will assume the burden of oxidation and reduction without adverse side effects.
LITERATURE CITED Giddings. J. C.: Yang, F. J. F.; Myers, M. N. Anal. Chem. 1974, 46, 1917. Giddings, J. C.; Caldwell, K. D.: Jones, H. K. I n Particle Size Distribution : Assessment and Charactefization: Provder, T., Ed.: ACS Symposium Series No. 332;Amerlcan Chemical Society: Washington, DC, 1987; Chapter 15. Giddings, J. C. Chem. Eng. News 1988, 66, 34. Kirkland, J. J.; Yau, W. W. Science 1982, 278, 121. Dalas, E.; Karaiskakis, G. Colloids Surf. 1987, 28, 169. Martin, M. I n Particle Size Analysis 1985; Lloyd, P. J., Ed.; Wiley: New York, 1987: pp 65-85. Oppenheimer, L. E.; Smith, G. A. Langmuir 1988, 4 , 144.
Anal. Chem. 1991, 63, 299-304 Caldwell, K. D. Anal. Chem. 1988, 60. 959A. Janca, J. FkH-Fbw fractbnatlon: Anaiysls of Macromoh?cules and Particles; Marcel Dekker: New York, 1988. Qiddings. J. C.; Yang, F. J.; Myers, M. N. J. Vlrol. 1977, 27, 131. W i n g s , J. C.; Lin, 0. C.; Myers, M. N. J. C o M Interface Sci. 1978, 65, 67. Gddings. J. C.; Chen, X.; Wahiund, K.-G.; Myers, M. N. Anal. Chem. 1987, 59, 1957. Barman, B. N.; Myers, M. N.; Giddings, J. C. Powder Techno/. 1989, 59,53. Wahlund, K.-G.; Litzb. A. J. Chromatcgr. 1989. 467, 73. Caidweii, K. D.; Karaiskakis, G.; Myers, M. N.; Glddings, J. C. J. Pharm. Sci. 1981, 70, 1350. Yonker, C. R.; Jones, H. K.; Robertson, D. M. Anal. Chem. 1987, 59, 2573. Thompson, G. H.; Myers, M. N.; Giddings, J. C. Anal. Chem. 1969, 4 7 , 1219. Giddings, J. C. I n Size Exclusion Chromatcgraphy; Hunt, B. J., HoMing, S . . Eds.; Blackie and Son: Glasgow, 1989; Chapter 8. Kirkland, J. J.; Rementer, S. W.; Yau, W. W. Anal. Chem. 1988, 6 0 , 610. Gao, Y. S . ; Caidwell, K. D.; Myers, M. N.; Glddlngs, J. C. Macromolecules 1985, 78. 1272.
299
(21) Schimpf, M. E.; Myers, M. N.; W i n g s , J. C. J . Appl. Polym. Sci. 1987, 33, 1170. (22) GLddings, J. C. Sep. Sci. Techno/. 1984, 79, 831. (23) Giddings, J. C.; Yang, F. J.; Myers, M. N. Anal. Blochem. 1977, 87, 395. Corresponding author.
Guangyue Liu J. Calvin Giddings* Field-Flow Fractionation Research Center Department of Chemistry University of Utah Salt Lake City, Utah 84112 RECEIVEDfor review July 3,1990. Accepted October 12,1990. This work was supported by Grant CHE-8800675 from the National Science Foundation.
TECHNICAL NOTES Vapor Sampling Device for Direct Short Column Gas Chromatography/Mass Spectrometry Analyses of Atmospheric Vapors Neil S. Arnold,* William H. McClennen, and Henk L. C. Meuzelaar Center for Micro Analysis and Reaction Chemistry, University of Utah, Salt Lake City, U t a h 84112
INTRODUCTION A number of methods are currently used for atmospheric vapor and gas sampling with mass spectrometric detection and identification. Direct mass spectrometry (MS) sample introduction methods include fixed molecular leaks (I), atmospheric pressure ionization ( 2 , 3 ) ,trap and desorb (4),and membrane separation (5), while gas chromatography/mass spectrometry (GC/MS) methods employ trap and desorb (6), direct bubbler solvent injection (7,8), sample loops (9), and pressurized gas plug introduction (10). Approaches vary depending upon whether MS, tandem MS, or GC/MS analyses are desired. Direct MS and tandem MS analyses typically give quick response times and high repetition rates but are often sensitive to interferents, including atmospheric constituents, while GC/MS analyses offer greater specificity but with typically slower results. The value of short capillary column GC/MS has been demonstrated for rapid analyses of many compounds ( I I , 12) including thermally labile and polar compounds (13). Recent work by Hail and Yost (14) has continued this trend. Such investigations have encouraged the use of a standard GC/MS transfer line on the mass spectrometer (designed primarily as a pressure drop from atmosphere to vacuum) as a short (l-m) capillary GC column for separation of atmospheric components. Further, the enhanced sensitivity capabilities of the ion trap mass spectrometer (Finnigan-MAT) (15,16), to detect sample quantities on the order of 100 fg ( I 7)in both full-scan MS and MS/MS modes, allow for direct sample introduction without preconcentration. The presently described direct atmospheric sampling inlet and methodology attempts to bridge the gap between the slow GC/MS and interferent-sensitive direct MS approaches by utilizing "transfer line" chromatography for a separation of sample components and ion trap mass spectrometery for rapid detection and identification.
EXPERIMENTAL SECTION A schematicdiagram of the vapor inlet system appears in Figure 1 (18). It comprises three concentric tubes whose internal flows
control the vapor sampling process. The innermost tube is the transfer line column (0.18- or 0.15-mm-i.d. fused silica capillary column) to the mass spectrometer. The intermediate tube consisted of 0.53-mm-i.d. deactivated fused silica capillary column (Supelco) cut to extend approximately 2 cm beyond the end of the inner tube. The outer tube was 3-mm-0.d. quartz or 1/8-in. glass-lined steel (GLT, SGE). All other fittings are either standard components of an ion trap detector (ITD 700, Finnigan-MAT) transfer line housing or are modified Swagelok fittings as shown in Figure 2. The width and frequency of the injection pulse onto the column are controlled by a computer, but the actual switching speed of the inlet is limited by compressible volumes and flow restrictions in the overall plumbing (including the valves). The system is t y p i d y run with 10-30 mL min-' of gas sampled through the large-diameter outermost tube. Between injections, 1-5 mL min-' of helium carrier gas is expelled from the intermediate-diameter (0.53-mm) tube into this flow in order to prevent atmospheric access to the capillary column. During the injection, the intermediate tube flow is reversed to provide direct atmospheric access to the head of the capillary GC column. Further, these flows are adjusted so that the flow into the large tube is unchanged by the sampling position., The transfer line capillary consisted of 1.1m of a 0.18-mm-i.d. fused silica capillary column coated with 0.4-pm DB-5 stationary phase (J&W Scientific)or a similar length of 0.15-mm-i.d. column coated with 1.2-pm film thickness CP SIL 5 CB (Chrompack). With a pressure drop from ambient (0.85 atm at 4400-ft elevation) to vacuum (approximately Torr), the flow rates were 4 mL min-' (270 cm s-') and 1.5 mL m i d (200 cm s-l), respectively, for the two capillary inside diameters at 25 OC. Transfer line isothermal operating temperatures ranged from 25 to 250 "C depending upon the target compound volatility. The inlet temperature was also varied for different target compounds and was typically operated more than 25 "C higher than the column temperature.
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