292
Anal. Chem. 1991, 63,292-296
(7) Failey. Michael P.; Anderson, David L.; Zoller, William H.;Gordon, Glen E.;Lindstrom, Richard M. Anal. Chem. 1979, 57, 2209-2221. (8) Sears, Varley F. Adv. Phys. 1975, 24, 1-43.
RECEIVED for review July 19,1990. Accepted October 29,1990. This work was supported in part by the National Institute of Standards and Technology through Grant/Cooperative Agreement No. 70NANB9H0903 to the University of Maryland. This work will be included in the dissertation to be submitted in partial fulfillment of the requirements for a Ph.D.
degree in Chemistry by E.M. at the University of Maryland. Certain commercial equipment, instruments, or materials are identified in this paper in order to adequately specify the experimental procedures. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. Contributions of the National Institute of Standards and Technology are not subject to copyright.
2-(9=Anthryl)ethyl Chloroformate: A Precolumn Derivatizing Reagent for Polyamines Determined by Liquid Chromatography and Fluorescence Detection A r t h u r J. F a u l k n e r a n d Hans Veening* Department of Chemistry, Bucknell university, Lewisburg, Pennsylvania 17837 Hans-Dieter Becker Department of Organic Chemistry, Chalmers University of Technology and University of Gothenburg, S-412 96 Gothenburg, Sweden
This paper describes the use of a new compound, 2-(9anthry1)ethyl chloroformate (AEOC), as a sensltlve and convenient precolumn, fluorophorlc, derlvatlrlng reagent for polyamlnes (putrescine, Cadaverine, spermldlne, and spermine) determined by llquld chromatography. Experiments were carried out to determlne maximum fluorescence excitation and emlsslon wavelengths, optlmum reaction pH, calibration curves, and mlnlmum detection llmlts for each of the AEOC-derlvatired polyamines. The procedure was applied successfully to serum samples from healthy lndlvlduals and cancer patlents and provides complete separatlon of derivatired polyamines from serum hydrolysate components. Detection llmlts for three of the AEOC-derlvatlzed polyamlnes are more favorable than those reported previously for polyamines derlvatlzed with 9-fluorenylmethyl chloroformate (FMOC).
INTRODUCTION Elevated levels of polyamines (putrescine, cadaverine, spermidine, and spermine) in human urine, serum, and tissue samples have been associated with the rapid regeneration or regrowth of tissue for some time. The analytical determination of these compounds is important for monitoring the clinical progress of cancer patients. Liquid chromatography (LC) using ion-exchange (1-3) or reversed-phase columns (4-8)has been used extensively to determine polyamines in biological fluids. Direct detection of these compounds is difficult, because they do not absorb in the ultraviolet (UV) and, consequently, do not possess native fluorescence. Therefore, LC procedures require either pre- or postcolumn derivatization techniques to produce detectable fluorophores.
* To whom correspondence should be addressed.
Previously, we reported the use of 9-fluorenylmethyl chloroformate (FMOC) as a precolumn derivatizing agent for determining polyamines by reversed-phase LC and fluorescence detection (6). Excitation and emission wavelengths in the case of these FMOC-derivatized compounds were 265 and 340 nm, respectively. FMOC has also been used for precolumn derivatization and LC separation of amino acids (9-11). In this paper we report the use of 2-(9-anthryl)ethyl chloroformate (AEOC) as a novel LC precolumn, fluorophoric reagent for polyamines. The absorption and emission spectral properties of the anthracene chromophore have previously been recognized to be analytically attractive features. Thus, as early as 1941, amino groups were reported to be spectrophotometrically accessible by reaction with 2-anthryl isocyanate (12). By the same token, isocyanates can be detected by derivatization with amino-substituted anthracenes, such as 9-((methy1amino)methyl)anthracene (13). During the past decade, a variety of anthracene derivatives, namely, 9anthryldiazomethane ( I 4), 9-(chloromethy1)anthracene(15), 1-anthroyl nitrile and 9-anthroyl nitrile (16),9-anthrylmethanol (10,p(9-anthroy1oxy)phenacyl bromide (It?), and 9-anthroyl chloride (19),have been applied in an analytical context. Moreover, optically active (1-(1-anthry1)ethyl)amine and (1-(2-anthryl)ethyl)amine have been used in the LC analysis of chiral carboxylic acids (20). As far as we know, anthryl-substituted chloroformates have not been reported as derivatizing agents in LC. Earlier attempts to prepare the chloroformate of 9-anthrylmethanol were unsuccessful insofar as this chloroformate spontaneously decomposed into 9-(chloromethy1)anthracene by elimination of carbon dioxide (21). By contrast, the chloroformate of 2-(9-anthryl)ethanol (AEOH) has been found recently to be easily accessible and stable in its crystalline state (22). Its preparation proceeds in a straightforward fashion by adding an ether solution of 2-(9-anthryl)ethanol(23)to a commercially available solution of phosgene in toluene at 0 OC wing pyridine as an HCl scavenger. AEOC forms colorless crystals (from
0003-2700/91/0363-0292$02.50/00 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
pentane) that melt a t 86-87 "C. In its UV spectrum in cyclohexane solution, absorption maxima (e, L mol-' cm-') are exhibited at 249 (91OOO), 256 (180000), 332 (3100), 348 (6600), 366 (llOOO), and 386 (11000) nm. Thus, the high molar absorptivity around 256 nm implies low limits of detection. For the fluorene chromophore, by contrast, the molar absorptivity a t 262 nm is only 19 400 L mol-' cm-'. Moreover, excitation in the long-wavelength region of the anthracene chromophore, around 386 nm, facilitates selective excitation of derivatization products in the presence of aromatic substrates absorbing in the short-wavelength region. The emission of the anthracene fluorophore allows for detection >400 nm. Fluorescence quantum yields of 2-(9-anthryl)ethanol, AEOC, and methyl 2-(9-anthryl)ethyl carbonate in cyclohexane solution are 0.27,0.23, and 0.21, respectively (22). Although the fluorene fluorophore in FMOC derivatives is characterized by a higher emission yield (0.54) ( 2 4 ) , the far larger molar absorptivity of the anthracene chromophore should lead to enhanced sensitivity for AEOC-derivatized amines. The validity of this assumption is indeed borne out in the HPLC determination of AEOC-derivatized amino acids (25).
EXPERIMENTAL SECTION Reagents and Chemicals. Putrescine (PUT), cadaverine (CAD), spermidine (SPD), and spermine (SPM) were obtained as the hydrochloride salts from Sigma Chemical Co. (St. Louis, MO 63178) and were used without further purification. Reagent grade sodium borate, hydrochloric acid, sodium hydroxide, potassium hydroxide, and potassium dihydrogen phosphate were obtained from Fisher Scientific Co. (Fair Lawn, NJ 07410). HPLC grade acetonitrile was obtained from J. T. Baker (Phillipsburg, NJ 08865). HPLC grade water was generated in-house from a filter unit produced by Industrial Water Technology (Attleboro, MA 02761). Bis(2-(9-anthryl)ethyl)carbonate (BAEC), 2-(9anthry1)ethyl chloroformate (AEOC), and 2-(9-anthryl)ethanol (AEOH) were synthesized as previously described (22,23). Since AEOC solutions have been found to undergo reactions upon exposure to laboratory light (26),reagent stock solutions were kept in light-protected containers and stored under refrigeration when not in use. Apparatus. The chromatographic system consisted of a tertiary SP-8700pump (Spectra-Physics, Santa Clara, CA 95051), supplied with a Model 7010 sample injector (Rheodyne, Inc., Cotati, CA 94928) and a 20-pL loop. The analytical column was a LiChrospher 100 RP-18,125 X 4.0 mm (id.), packed with 5-pm particles (EM Science, Cherry Hill, NJ 08034). Fluorescence detection was achieved with a Model GM-970 instrument (Schoeffel, Division of Kratos, Westwood, NJ 07675). The sensitivity range was set at 1.0 pA with a time constant of 1.5 s and high suppression. An excitation wavelength of 258 nm (5-nm bandwidth) and a 418-nm low cut-off emission fiter (Kratos) were used. Fluorescence spectra were obtained with an AmincoBowman 54-8961spectrofluorometer and a Houston Omnigraphic X-Y recorder (Houston Instrument Co., Austin, TX 78700). All chromatograms were recorded with a Model SP-4270 integrator (Spectra-Physics). Samples were separated at a flow rate of 1.0 mL/min with a binary gradient of 80% acetonitrile/20% DI water (pH = 6.1) to 100% acetonitrile in 10 min, with an isocratic post time of 15 min. The initial and final column inlet pressures were 480 and 330 psi, respectively. The column temperature was ambient. Precolumn Derivatization. Polyamine standards were derivatized by adding 100 pL of pH 9.0 borate buffer (0.025 M) and 500 pL of 0.12 mM AEOC in HPLC grade acetonitrile to 400 pL of a standard polyamine-containing (5 pg/mL) sample. The derivatization mixture was allowed to stand in the dark at 36 "C for 5 min. For some experiments,a postderivatization, preinjection dilution was performed by using a 1:l mixture of HPLC grade acetonitrile and water. Serum samples required a modified procedure because of the hydrolysis step preceding derivatization. Hydrolyzed serum (100 pL) was derivatized with 400 pL of pH 9.0 borate buffer (0.025 M) and 500 pL of 5.8 mM AEOC stock solution. In certain instances, the effluent from the column containing the desired fraction was collected for further
V1d;\ 200
300
293
I
I
I
400
500
800
700
Wavelength (nm)
Figure 1. Fluorescence excitation and emission spectra of eluted AEOC-PUT (0.5 pg of PUT with excess AEOC injected).
fluorescence spectral studies. This was done by derivatizing 0.5 pg of each polyamine with excess AEOC in borate buffer and collecting the eluted derivative in five successive runs, for the duration of full-scale detector response. Preparation of Samples. Serum samples from normal and cancer patients were obtained from Evangelical Community Hospital, Lewisburg, PA, and Geisinger Medical Center, Danville, PA, respectively. Normal samples were pooled from five individual specimens (2-3 mL), whereas cancer serum samples were treated without pooling. Serum samples were stored at -25 OC, when not in use. It was found necessary to hydrolyze serum with HC1 prior to analysis in order to release the polyamines from the conjugates that they form with proteins and other components in serum. The procedure consisted of a 10-h reflux of 5.0 mL of serum with an equal volume of 6 M HC1 (6). The resulting hydrolysate was evaporated to dryness under vacuum at a temperature of 50 "C. The residue was neutralized with dilute KOH, reevaporated and diluted with HPLC grade water to a volume of 5.0 mL. The resulting solution was next centrifuged for 10 min at 3400 rpm and filtered through a 0.45-pm Millex-HV filter unit (Millipore). Spiked serum samples, needed for recovery studies, were prepared by adding a known amount of each polyamine prior to hydrolysis.
RESULTS AND DISCUSSION Derivatization of primary and secondary amines with AEOC results in the formation of fluorescent urethanes. A general reaction between AEOC and a primary amine is shown in eq 1. It is likely that AEOC derivatizes with both primary amine
TYN Fyc, +
AEOC
0
/
/
/
+
HCI
(1)
RNHp
0
groups of the polyamines and that further derivatization may occur with the secondary amine groups in spermidine (one) and spermine (two). Figure 1illustrates the fluorescence excitation and emission spectra for five collected fractions of the PUT-AEOC derivative. Similar spectra were recorded for AEOC and AEOH. The emission band for polyamineAEOC derivatives is located in a higher wavelength region (380-450 nm) than that reported previously (6) for polyamineFMOC derivatives (280-380 nm). On the basis of the spectra obtained, HPLC detection of polyamine-AEOC derivatives was optimized by using an excitation wavelength of 258 nm and a 418-nm low cut-off emission filter. Use of a 390-nm low cut-off filter was investigated and found to be much less sensitive than the 418-nm filter. It a p p e m that the AEOC derivatives of polyamines are very stable, as evidenced by the fact that the derivatized fractions that were collected, showed very little degradation when analyzed by HPLC after several days of standing at room temperature.
294
ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
1
1
AEOH
'21
7
7.5
1
1.5
a
a.s
Derivatization pH
10
10.s
Figure 2. Detector response vs derivatization pH for eluted AEOCpolyamine derivatives (0.2 ng of each polyamine injected).
m Y
CAD
I PUT
Time (min) 25 Figure 4. Chromatogram for AEOCderivatized polyamine standards (0.2 ng of each polyamine injected). Conditions: same as in Figure 0
3.
by derivatizing each of the four polyamines a t pH values ranging from 7.0 to 10.5 and measuring the fluorescence response (Aexc = 258 nm; A, = >418 nm) for each eluted analyte as a function of reaction pH. From these results, shown in Figure 2, an optimum derivatization pH of 9.0 was selected for all subsequent experiments. At lower pH values, it is possible that excessive polyamine protonation prevents complete derivatization; whereas a t pH > 9, the hydrolysis of AEOC appears to be favored. Dry (0.003% HzO) HPLC grade acetonitrile was chosen as the AEOC reagent solvent because there was no observable hydrolysis of AEOC to AEOH. Acetonitrile was also convenient because of its complete miscibility with aqueous polyamine samples during the derivatization step and subsequent chromatographic separations. A chromatogram for AEOC reagent solution is shown in Figure 3. The identity of two of the impurity peaks, AEOH and bis(Z-(g-anthryl)ethyl) carbonate (BAEC), was c o n f i i e d by comparison of their retention times to those of authentic samples that were independently synthesized. The reaction of AEOC with AEOH to form the carbonate (BAEC) is illustrated in eq 2. Neither of these byproducts interfered in
AEOC
I :: I
0
BAEC
Time (min)
25
Figure 3. Chromatogram for 0.13 ng of AEOC in acetonitrile. Con-
ditions: flow rate, 1.O mL/min; ambient temperature; column, LiChrospher 100 RP-18, 125 X 4.0 mm; detection, fluorescence (k,,, = 258 nm, A,, > 418 nm). Complete derivatization of polyamines with AEOC was found to require 30 min a t ambient temperature. At 36 OC, however, a reaction time of only 5 min was needed in order for quantitative conversion to occur. Derivatization at higher temperatures (40, 50, and 60 "C) offered no additional time-saving advantage. As AEOC reagent was always used in large excess, derivatization of all primary and secondary amino groups is virtually ensured. The observed retention times for AEOC-derivatized polyamines also indicate that all primary and secondary amino groups have been protected. The reaction of AEOC with polyamines was also found to be pH dependent. The optimum reaction pH was determined
vyc, q 3+
0 AEOC
AEOH
BAEC
the separation of the polyamines; in fact, BAEC was found to be a useful "relative retention time" internal standard. A chromatogram for the separation of a standard mixture of the four polyamines is shown in Figure 4. Although, the
ANALYTICAL CHEMISTRY, VOL. 63,NO. 3, FEBRUARY 1, 1991
295
Y)
%
PUT I:,
BAEC
rx,
2 X
8L "
a
Y 0 "
a
e -0
3
1s
0
a
L L
0 0 10
-
o!
0.0
v 0.1
,
0.2
0.S
0.4
0.5
0.6
0.7
1
0.0
ng PoLyomine Injected Flgure 5. Corrected peak area vs nanograms of polyamine injected.
(Experimental data replotted according to ref 27.)
Time (min)
25
Table I. Minimum Detection Limits and Quantitative Reproducibility for Polyamine-AEOC Derivatives
detection limitsa compd limit of detection, pg of free base PUT CAD SPD SPM a 33
1.3 1.1
1.5 9.9
reproducibilityb no. of samples coeff of analyzed variation 5 5 5 5
IAEC
0.5 0.7 0.7 1.2
pg of polyamine injected. b0.2 ng polyamine injected.
retention times varied slightly from run to run, the retention times relative to BAKU were very consistent. Under the gradient conditions used, the four polyamine-AEOC derivatives were completely resolved from each other and from excess reagent and reaction byproducts. A plot of corrected peak area versus nanograms of polyamine injected is shown in Figure 5. Data points were obtained for 0.05, 0.10, 0.25, 0.50, and 0.75 ng of polyamine injected. The original plots showed significant curvature due to nonlinear detector behavior. In order to show the response linearity more clearly, the data were replotted according to the method of Dorschel et al. (27). The limits of detection and reproducibilityof the procedure were determined for each of the four polyamines, as shown in Table I. In this procedure, 33 pg of each polyamine free base was injected while the detector was set at the lowest attenuation. The minimum detection limit was calculated by using a signal to noise ratio (S/N) of 3 to 1where the signal value (S)used was peak height of the polyamine and the noise (N) was the width of the baseline when no peaks eluted. The detection limits determined for AEOC-derivatized PUT, CAD, and SPD (1.3, 1.1,and 1.5 pg, respectively) are more favorable than those reported previously (6) for polyamines derivatized with FMOC (4,9, and 7 pg, respectively). The SPM limit was found to be slightly higher (9.9 pg). This provides more than sufficient sensitivity for clinical use. The reproducibility of the procedure was investigated by making several consecutive injections of 0.2 ng of each polyamine. The
Time (min)
is
Figure 6. (A) Chromatogram for hydrolyzed normal serum. (6)
Chromatogram for hydrolyzed cancer serum. Condftions: same as in Figure 3. integrated area for each polyamine was averaged from each of the runs. Figure 6 illustrates chromatograms for hydrolyzed, AEOC-derivatized, normal (A) and breast cancer (B) serum samples. In the normal serum sample (A), the only polyamine found was PUT (0.70 mg/100 mL of serum). In the cancer serum sample (B), the PUT concentration was found to be considerably elevated (3.0 mg/100 mL of serum). A second cancer serum sample showed a PUT concentration of 14.0 mg/100 mL serum and traces of CAD and SPD. Replicate determinations of five cancer serum samples indicated satisfactory reproducibility (CV = k0.93). The retention times of the polyamines were confirmed by analyzing the same cancer serum samples, spiked with 0.9 pg of each polyamine,
296
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, 5 8 , 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
