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Anal. Chem. 1982, 5 4 , 948-950
(11) Smillie, R. d.; Sakuma, T.; Duholke, W. K. J. Environ. Scl. Health, Part A 1978, A 13, 187. (12) Symons, J. M.; Bellar, T. A.; Carswell, J. K.; DeMarco, J.; Kropp, K. L.; Robeck, G. G.; Seeger, D. R.; Slocum. C. J.; Smith, B. L.; Stevens, A. A. J. Am. Water Works Assoc. 1975. 8 7 , 634. (13) Grote, J. 0. Am. Lab. (Fairtleld, Conn.) 1975, 7 , 47. (14) Burgett, C. A.; Green, L. E. “Application Note ANGC 5-78”; HewlettPackard Co.: Avondale, PA, 1976. (15) Daemen, J. M. H.; Dankelman, W.; Hendrlks, M. E. J. Chromatogr. Sci. 1975, 13. 79. (16) Otson, R.; Wllliams, D. T.; Bothwell, P. D. Environ. Scl. Technoi. 1979, 13, 936. (17) Nicholson, A. A.; Meresz, 0.; Lemyk, B. Anal. Chem. 1977, 49,814. (18) Thomason, M.; Shoults, M.; Bertsch, W.; Holzer, G. J. Chromatogr. 1978, 158,437. (19) Qulmby, B. D.; Delaney, M. F.; Uden, P. C.; Barnes, R. M. Anal. Chem. 1979, 51,875. (20) “The Canada Gazette Part I ” , Supply and Servlces Canada: Ottawa, Ontario, Canada, 1979, Dec 1; p 7365.
(21) “Guidelines for Canadian Drlnking Water Quality 1978”, Health and Welfare Canada: Ottawa, Ontario, 1978. (22) Keith, L. H.; Telliard, W. A. Environ. Sci. Technol. 1979, 13, 416. (23) Otson, R.; Williams, D. T. J. Chromatogr. 1981, 212, 187. (24) Malalyandl, M.; Sadar, M. H.; Lee, P.; O’Grady, R. Water Res. 1980, 14, 1131. (25) Otson, R.; Williams, D. T.; Bothwell, P. D. J. Assoc. Off. Anal. Chem., In press. (28) Garrlson, A. W. Ann. N.Y. Acad. Scl. 1977, 298,2. (27) Cotruvo, J. A.; Wu, C. J. Am. Water Works Assoc. 1978, 7 0 , 590. (28) Shackelford, W. M.; Keith, L. H. “Frequency of Organic Compounds Identified in Water”, EPA-600/4-78-062, U S . Environmental Protection Agency: Athens, GA, Dec 1976. (29) Fed. Regist. 1979, 44, 69468. (30) Kaiser, R. J. Chromatogr. Sci. 1971, 9 ,227.
RECEIVED for review June 1,1981. Accepted December 28, 1981.
Sequentially Excited Fluorescence Detection in Liquid Chromatography Paul B. Huff, Bruce J. tromberg, and Michael J. Sepaniak” Department of Chemistry, Universi@of Tennessee, Knoxville, Tennessee 379 16
A fluorometrlc detection technique for liquld chromatography Is descrlbed, whereln fluorescence is monitored from a highly excited molecular state following sequentlai resonant excitation using a tunable laser source. Low nanogram minimum detectable lnjectlon quantlties and linearity over 3 decades of concentratlon are demonstrated in the separatlon of derlvatized amlnes and the antltumor drugs adrlamycln and daunorublcln. Improvements in spectral selectivity and sensltlvlty which would result from uslng state of the art laser and signal recovery equlpment are dlscussed. The highly colllmated laser output facllltates the mlnlaturlzatlon of flow cells. The dependence of fluorescence signals on photon density, for techniques involving excltatlon with two photons, Is shown to permlt mlniaturlratlon of flow cells without loss In signal.
Characterized by excellent sensitivity and selectivity, fluorimetry has been used considerably in liquid chromatography detection. A recent review article on laser fluorometric detection in liquid chromatography (I)emphasized that lasers, with their unique spectral characteristics, offer important advantages over incoherent sources for excitation of molecular fluorescence in liquid chromatography detection. Among the most notable accomplishments of laser fluorometric detection in liquid chromatography are minimum detectable injection quantities in the low to subpicogram range (2-4), the development of flow cells with submicroliter volumes ( 4 , 5 ) ,and the use of pulsed lasers in time-resolved fluorimetry to enchance selectivity for polycyclic aromatic hydrocarbons (6). The high output power of laser sources also makes it possible to observe a number of “nonlinear” spectroscopic phenomena under conditions realistic to liquid chromatography detection. Coherent anti-Stokes Raman detection has been employed by Carreira and Rogers in a computer-controlled liquid chromatographic system (7)and Sepaniak and Yeung have used two-photon excited fluorescence (TPEF) in the liquid chromatographic separation of certain oxadiazole
molecules (8). With two-photon excitation a resonant transition in a molecule is accomplished by the simultaneous absorption of two photons. The principal analytical advantage of the two-photon excitation process is that it involves different selection rules than the more conventional one-photon excitation process (9) and this provides an added dimension for spectral selectivity in fluorometric detection. This advantage is also exhibited by the sequentially excited fluorescence (SEF) detection described in this report. The SEF process is diagramed in Figure 1. A symmetric singlet ground state of an organic molecule is coupled, via two resonant absorptions (Le., So + hul S1* + hv2 Sn*), to a highly excited singlet state with the same symmetry. This is the opposite excited state symmetry as that observed in a centrosymmetric molecule for a one-photon excitation. Although the greatest analytical selectivity for SEF will result from using two independently tunable excitation sources, the work presented herein was performed with a single laser excitation source. With SEF, fluorescence (If,,) is monitored from a highly excited singlet state, as opposed to the SI*state as in conventional fluorescence or TPEF. Internal conversion to an intermediate state of unsymmetric character is shown in Figure 1. This added restriction for the SEF process results from the fact that direct emission from the symmetric S,* state to the symmetric ground state is forbidden. Actually, it is not necessary for the intermediate state to belong to an electronic state other than Sn*, since it can acquire the necessary symmetry through vibronic coupling with other electronic states. The unique selectivity of SEF is of very limited value if concentrations meaningful in liquid chromatography cannot be detected. Because of the proximity of electronic states above the first excited state, and vibronic coupling between these states, internal conversion to the SI* state, and not fluorescence,is by far the most efficient means of depopulating highly excited states. This is consistent with Kasha’s rule, which states that “the emitting electronic level of a given multiplicity is the lowest excited level of that multiplicity (IO).” However, the rate constant for internal conversion has an
0003-2700/82/0354-0946$01.25/00 1982 Amerlcan Chemical Society
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-
s;l(+-;
SiWI-
E: (u)
sgw--
Flgure 1. Electronic energy level diagram for the SEF process.
upper limit equal to typical molecular vibrational frequencies (about s-l). Radiative rate constants for highly allowed transitions are in the los to lo9 range. Accordingly, fluorescence quantum efficiencies for highly allowed transitions from highly excited states have a lower limit of about lo4 to Nevertheless, extremely low background levels associated with sequential excitation (vide infra) permit detection of weak fluorescence from highly excited states. For instance, Lin and Topp have obtained sequentially excited fluorescence spectra of certain aromatic hydrocarbons, a t lo-&M concentrations, using a N2-pumped dye laser for excitation (11). An expression for the intensity of SEF signals can be derived by using kinetic equations which relate the number of higher excited state fluorescence photons to various experimental parameters and electronic transition rate constants. The rate constant for depopulation of S*, is given by where kf, kx, and ki, are the individual rate constants for fluorescence, intersystem crossing, and internal conversion, respectively. When n is greater than one, ki, usually dominates the other two terms. For the case when a single continuous wave laser, not sufficiently intense to saturate excited states, is used for both the So to S1* and SI*to S*, transitions, the steady-state rate expressions for the populations of S1* and S*, can be solved to yield
where If,m i the SEF intensity, K is an instrumental efficiency factor, P is the laser power, A is the cross-sectional area of the focused laser beam, ii is a sequential absorption coefficient, b is path length, and C is concentration. Equation 2 can be mlodified for pulsed excitation, using lasers with relatively large pulse widths, by multiplying by the duty cycle of the h e r . However, the steady-state approximation used in deriving eq 2 is not valid for the case of molecules with first excited state lifetimes ( l / k d , ) comparable to the exciitation pulse width. In those situations a transient treatment should be employed. In the extreme case of excitation pulse widths significantly less than the first excited state lifetime (e.g., using synchronously pumped dye lasers for excitation) the SEF signal is dependent on the excitation pulse energy, as opposed to the excitation pulse power, and independent of k d l .
EXPERIMENTAL SECTION For mo8t of this work the laser radiation was focused with a
25-mm focal length lens into a simple 1.0 mm i.d. X 2.0 mm o.d., Suprasil quality quartz, capillary tube flow cell. The flow cell was supported and positioned with a Newport Research Corp., Fountain Valley, CA, Model FP-1 fiber-optic positioner and the blue-shifted fluorescence emission was collected at a 90° angle with a f / l quartz lens. Excitation was provided by either a Nz-pumped dye laser (National Research Group, Madison, WI, Model NRG-0.5-5-150/B Nz laser and Model NRG-DL-0.03 dye laser) tuned to 488 nm using Coumarin 481 dye or an argon ion laser (ControlLaser, Orlartdo,FL, Model 553, or Spectra Physics,
ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982
947
Mountain View, CA, Model 171) operating a 488 nm. The Nzpumped dye laser was operated at 60 Hz with an average power of 8 mW (peak power approximately 25 kW). The continuous wave argon ion lasers were operated at 1-3 W. A Corning GG-455 sharp cutoff filter was used to block laser plasma lines or superradiance at wavelengths shorter than the excitationwavelengtlh. A combination of a saturated CuS04 solution and three Corning 7-54 band-pass fiikrs was used to isolate the blue-shiftedemission. SEF signals for the separation of the antitumor drugs adriamycin (A,) and daunorubicin (D,) were detected with an Amperex 56 DVP photomultiplier tube (PMT) operated at 2000 V and processed with an Ortec, Oak Ridge, TN, photon counting system. Several alkyl and aromatic amines were derivatized with '7chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) by adding a weighed amount of the amine to a methanol solution containing a 10-fold excess of NHD-C1. SEF signals for these derivatized amines were detected with an RCA 1P21 PMT operated at 900 V and processed with a Pacific Precision Instruments, Concord, CA, Model 126 quantum photometer. The quantum photometer was operated in the photon counting mode when argon ion laser excitation was used and in the nanoampere current measuring mode when Nz-pumped dye laser excitation was used. Pulsed laser excitation produced signal pulses that were partially shunted to ground by a diode clamp at the electrometer input of the quantum photometer. To minimize this problem, we increased the feedback capacitance for the current measuring operation(a1 amplifier of the electrometer by a factor of 10. This slowed the electrometer response but did not noticeably distort chromatographic peak profiles. The chromatographicsystem was composed of a reciprocating piston pump (LaboratoryData Control, Riviera Beach, FL, Model 196-0066-001 minipump or Altex, Berkeley, CA, Model 110), a Rheodyne, Berkeley, CA, Model 7010 fixed loop (20 wL) injector, and an Alltech (Arlington Heights, IL) 10 wm, 4.6 mm X 25 cm, C18chromatographic column. The antitumor drugs adriamycin (A,) and daunorubicin (D,) were supplied by Nicholas Bachur of the Baltimore Cancer Research Center and separated using an eluent that was 50% acetonitrile (HPLC grade) in 0.01 M phosphoric acid solution. The derivatized amines were separated with a gradient stepped from 60% to 75% methanol (HPLC grade) in water at 10 min into the separation. The flow cell miniaturization study was performed by using a dilute solution of the scintillator PBD. Excitation at 488 nm was provided by the Spectra Physics argon ion laser operating at 1.0 W. The half-angle divergence of this laser is specified as 0.56 mrad. TPEF emission from the PBD solution was isolated by use of the same optical filters reported above. A variety of glass or quartz, plano-convex or double-convexlenses with focal lengths ranging from 18 to 200 mm were used in this study. Laser ~ into one of three quartz, capillary tube flow radiation w a focused cells (1.0 mm i.d. by 2.00 mm o.d., 0.50 mm i.d. by 1.0 mm o.d., or 0.25 mm i.d. by 0.50 mm 0.d.). A signal was recorded for each lens-flow cell combination, after suitable optimization of the optical alignment.
RESULTS AND DISCUSSION For determination of its ultimate value in liquid chromatography detection, the advantages, differences, and limitations of SEF detection are compared to the conventional fluorescence technique (Le., one-photon excitation, S1* So emission). The following discussion will be presented in terms of the usual analytical parameters of sensitivity, linearity of response, and selectivity. Sensitivity. As seen from eq 2, SEF signals are quadratically dependent on excitation light intensity. Thus, the inherent inefficiency of the SEF process can be overcorine through the use of high-powered lasers. While increases in excitation light intensity can lead to more intense conventiorial fluorescence signals, improvements in detectability may not be realized due to a concomitant increase in optical background levels. Chromatographic base line noise is generally a function of the optical background. The large blue shift between excitation and emission wavelengths with the SEF technique enables one to spectrally reject stray radiation that
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948
ANALYTICAL CHEMISTRY, VOL. 54, NO. 6, MAY 1982 a
r
.
, 10
2'0
1
RETENTION TIME (minl
Chromatogram of the antitumor drugs adriamycin (A,) and demonstrating the sensitivity of SEF detection. Laser daunorublcin (0,) excitation was 2.0 W at 488 nm from an argon ion laser; 41 ng of each drug was injected. Flgure 2.
contributes to the optical background. Background levels near the dark currents of PMTs have been observed with the lasers previously described. Consequently, increases in signal levels which result from increasing the excitation light intensity are more likely to result in improvements in detectability with the SEF technique. Of course at some point nonlinear processes in the sample will produce stray radiation which cannot be spectrally resolved and even SEF detection will become background limited. The main source of optical background in laser fluorometric detection is generally the fluorescence of eluent impurities. In these situations an extremely powerful laser could result in similar background limited detectabilities for SEF detection and conventional fluorescence detection, providing thermal distortion problems are not encountered. This prediction is reinforced by the fact that large photon fluxes are less likely to saturate the emitting state in the SEF process (S,*) than the emitting state in the conventional fluorescence process (SI*). Typical SEF detection limits when moderate power continuous wave lasers are used for excitation are in the low nanogram range. This is demonstrated for the separation and SEF detection of the antitumor drugs adriamycin and daunorubicin in Figure 2 (12). Because of its quadratic power dependence, most high peak power, pulsed lasers should be better suited than continuous wave lasers for excitation in SEF. An example of this is illustrated in Figure 3 for the separation of the NBD derivatives of several amines. In this chromatogram the response for the last peak, the NBD derivatized n-octylamine,is shown for the case of (f) continuous wave excitation with an argon ion laser operating at 488 nm and (8) pulsed excitation with a N2-pumped dye laser (Coumarin 481 dye tuned to 488 nm). The ratio of peak height to chromatographic base line noise for this peak is slightly better for the pulsed laser case despite the fact that the average power for the Nz-pumped dye laser was about 200 times smaller than that of the argon ion laser. Moreover, the usual procedures for minimizing background with low duty cycle, pulsed laser excitation, such as gating the detector and cooling the PMT, were not employed. State of the art excimerpumped or Nd:YAG-pumped dye lasers are capable of peak powers and average powers about 10'-lo2 higher than the N2-pumped dye laser used in this work. SEF signals should significantly increase when these lasers are used for excitation. The magnitude of this increase depends on the amount of thermal distortion and excited-state saturation encountered, and will probably be less than the 102-104increase predicted by eq 2. Since the background observed for SEF detection in this work was primarily the continuous PMT dark current,
I
'
i0
'
2'0
3'0
1
RETENTION TIME lminl
Flgure 3. SEF chromatogram of the NBD derivatives of diethylamine (a),aniline (b), cyclohexylamine (c),di-n-propylamine (d), an unknown impurity (e), and n-octylamine (f) using argon ion laser excitation. The response for the n-octylamine derivative using N,-pumped dye laser excitatlon (9) is also shown. Roughly 0.5 pg of each derivative was injected.
detectability should also be improved by gating the detector and cooling the PMT. The SEF process being studied here was verified with two simple experiments. First, the effect of varying the argon ion laser power on chromatographic peak heights was observed. Near quadratic dependence of peak height on laser power was observed for all the compounds separated in this work, providing dilute solutions were injected. Second, the SEF emission was monitored after passing through the same optical, sharp cutoff fiiter used as an excitation fiiter. Absorption spectra for the antitumor drugs and derivatized amines show bands in the 450-510 nm range. These compounds also fluoresce (conventionalred-shifted emission) when excited at 488 nm. In all cases the SEF signal was completely removed by the filter, indicating that the signal was not conventional fluorescence leaking through the emission filters. Linearity of Response. The small dimensions of most liquid chromatography flow cells tend to minimize problems with preabsorption and postabsorption, two effects which can limit the linear dynamic range in fluorometric analysis. Nevertheless, even in liquid chromatography detection, if a significant amount of the excitation radiation is absorbed, the simple linear dependence of fluorescence signal on sample concentration will be lost. In the case of SEF detection the linear dynamic range is limited by thermal distortion of the laser beam, which results when a large amount of absorbed excitation energy is nonradiatively dissipated. The thermal distortion can increase the cross-sectional area of the focused laser beam, resulting in a decrease in the SEF signal as predicted by eq 2. Sample heating effects generally depend on the average power of the excitation source and, consequently, less thermal distortion would be expected for excitation with a low average power, high peak power, pulsed laser. This was visually confirmed by observing the distortion of the laser beam on a screen behind the flow cell as a concentrated solute band passed through the flow cell. The observed beam distortion was less for excitation with the N2-pumpeddye laser than for excitation with the argon ion laser. However, when pulsed lasers are used for excitation, linearity can be limited by detector saturation. The SEF signal is for the most part temporally coincident with the laser pulse. The N2-pumped dye laser used in this work had a pulse width of about 5 ns. Typical PMT operating bandwidths are on the order of 1GHz.
ANALYTICAL
a
3
2
I I 2
3
4
LOG NANOGRAMS NANOGRAMS INJECTED INJECTED
Figure 4. Calibration plots for NBD-cyclohexylamine using argon ion laser excitation
(a) and N,-pumped
dye laser excitation (b).
Thus, PMT saturation would be expected to limit the linear dynamic range to a few decades of concentration. In Figure 4 calibration plots for SEF detection of NBD derivatized cyclohexylamine illustrate the linear dynamic range obtained with the equipment used in this work. Selectivity. There are eieveral characteristics of SEF which influence the ability of the technique to spectrally distinguish coeluted compounds. Differences in excited-state symmetry requirements for sequential, two-photon excitation, when compared to conventional, one-photon excitation, are described above. The fact that these differences can lead to dissimilar conventional fluorescence and two-photon excited fluorescence chromatograms has been demonstrated for the separation of coal liquification samples (13). A large value of k f , is vital to conventional fluorescence since it translates into a large So to S1* absorption coefficient and a large fluorescence quantum e€ficiency. A large S1* population is important in SEF. This can be accomplishedwithout a large value of k , since the small Soto S1* absorption coefficient that results from a small k , can be compensated for by a long S1* lifetime. Thus, the prediction can be made that molecules with na* lowest excited singlet states, which typically have small Soto !gl* absorption coefficients and long S1* lifetimes, could give relatively large SEF responses. The ability to tune both excitation and emission photons is an often otated advantage in analytical selectivity of fluorometric analysis. SEF provides additional selectivity due to the availabitity of three resonant optical transitions which can be individually tuned with the technique. This requires two tunable laser beams which must be accurately overlapped in the flow cell. The analytical selectivity of the SEF technique is also influenced by the short lifetime of the emitting state. On the negative side, this short lifetime makes it impossible to use time-resolved fluorimetry as a means of distinguishing between coeluted compounds. However, on the positive side, the emission is essentially coincident with the S1* to S*, absorption. As a result molecular motion will not be as likely to scramble the polarization information present in that transition. For example, distinctive sequential excitation polarization spectra have been obtained for certain xanthene dye solutions (14). It should be relatively simple to experimentally obtain this type of polarization information in liquid chromatography detection and this should aid in distinguishing coeluted compounds. Current instrumental limitations in our laboratory have made it impossible to exploit the advantages in analytical selectivity of the SEF technique outlined in the previous paragraph. Future work involving excitation with two independent laser sources and the employment of an efficient polarizer and a sensitive, UV responding PMT are being
CHEMISTRY, VOL. 54,
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planned to investigate these areas. Flow Cell Miniaturization. One of the more promising areas of research in liquid chromatographyis the development of “microscale”columns (15). These columns have achieved, or possess the potential for achieving, very high chromatographic efficiencies. One bothersome feature of “microscale” columns is that they must be matched with ultralow volume detectors. In certain cases it would be necessary to reduce detector flow cell volumes to less than 10 nL before chromatographic efficiency is column, and not detector, limited (16). The highly collimated laser output facilitates the development of ultralow volume, optical detectors. However, most optical detector signals depend on the optical path length and at some point, even with laser light sources, the optical path length must be sacrificed in the miniaturization of flow cells. An inspection of the focusing properties of Gaussian laser beams reveals an interesting aspect of fluorescence techniques involving excitation with two photons (17). The cross-sectional area ( A ) of a focused Gaussian laser beam is approximately given by
(3) where f is the focal length of the focusing lens and 0 is the half-angle divergence of the laser beam. It is possible to maintain a relatively uniform focused cross-sectionalarea over. a distance designated the Rayleigh range ( 2 ) and given by z = A/X
(4)
where X is wavelength. SEF and TPEF signals depend in-. versely on the cross-sectional area of the laser beam in the flow cell. These signals should increase with decreasing f o cusing lens focal length (i.e., signal a l/f“), until the Rayleigh range is comparable to the optical path length of the flow cell. Further decreases in the focusing lens focal length will not, necessarily increase the signal since the average cross sectionall area of the laser beam in the flow cell is no longer related to the focal length in a simple manner. Furthermore, the ability to cleanly pass the laser beam through the flow cell is seriously hampered when the Rayleigh range is less than the opticall path dimension of the flow cell. It should be possible to reduce the optical path length in SEF and TPEF detectors, without, loss in signal, by simply using a shorter focal length lens. If chosen properly the shorter focal length lens results in EL smaller laser beam cross-sectional area which compensates for the decrease in optical path length. It was decided to use the Spectra Physics argon ion laser in a study of the effect of focal length and flow cell size on signal levels. This decision was based on the superior beam quality of that laser and the absence of detector saturation with continuous wave laser excitation. Although SEF and TPEF have the same dependence on laser power density (i.e., signal a P 2 / A ) ,they differ in that thermal distortion of the laser beam can occur with the SEF technique. This is particularly true with continuous wave laser excitation. In order to eliminate problems with thermal distortion, we performed this study by using a dilute PBD solution. PBD gives M relatively large TPEF signal when excited at 488 nm. The results of the study are shown in Figure 5. A linear increase in signal with the inverse square of the focusing lens focal length is observed for each of three flow cells used. Signals reach a maximum when the Rayleigh range is about 20% larger than the flow cell outer diameter. The maximum signals were approximately equal for the three flow cells. A, decrease in signal with shorter focal lengths is persumably due to difficulties with cleanly focusing the laser beam through the cylindrically shaped flow cells. The miniaturization of flow cells in SEF and TPEF detection is limited by the divergence of the laser beam and the
950
Anal. Chem. 1982, 54, 950-953
orders of magnitude poorer than conventional fluorescence. This limits its current value in real analysis, but it is likely that state of the art pulsed laser and signal recovery equipment will make the sensitivity of the technique more competitive, especially when separations are performed on “microscale” columns.
2.5
LITERATURE CITED
z e
u u
2 1.5
45
.3’5
,215
L O G 11/f21
Flgure 5. Logarithmic plot of the two-photon excited fluorescence signal of a dilute PBD solution vs. the inverse square of the focusing lens focal length ( f ) . Flow cell dlmensions a r e (a) 1.0 mm i.d. by 2.0 mm o.d., (b) 0.50mm 1.d. by 1.0 mm o.d., and (c) 0.25 mm i.d. by 0.50 mm 0.d.
availability of lenses. In the case of SEF, thermal problems may also be a factor. Nevertheless, we predict that optical path lengths could be reduced by at least an order of magnitude, versus what is normally used in conventional fluorescence,without loss in signal, thereby making the sensitivity of SEF and TPEF more competitive with conventional fluorescence detection, when used with “microscale” columns. In summary we have presented a laser-based detection technique for liquid chromatography which tends to probe different excited states than conventional optical detectors, In addition, the technique has a higher degree of tunable selectivity. The demonstrated sensitivity of SEF is about 3
(1) Yeung, E. S.;Sepaniak, M. J. Anal. Chem. 1980, 52, 1465 A-1470 A. (2) Dieboid, G. J.; Zare, R. N. Science 1877, 796, 1439-1441. (3) Sepaniak, M. J.; Yeung, E. S. J . Chromatogr. 1980, 790, 377-363. (4) Folestad, S.; Johnson, L.; Josefsson, B. I n “Proceedlngs of the Fourth Internatlonal Symposium on Capillary Chromatography”; Kaiser, R. E., Ed.; Instltute of Chromatography: Bad Durkheim, West Germanv. 1981; pp 405-427. (5) Hershberger, L. W.; Callis, J. B.; Christian, G. D. Anal. Chem. 1879, 57. 1444-1446. (6) Deutscher, S.B.; Richardson, J. H.; Clarkson, J. E.; Ondov, “Abstracts of Papers, 182nd National Meeting of the American Chemical Society, New York, NY, Aug, 1961; Amerlcan Chemical Society: Washington, DC, 1961; ANYL 48. (7) Carreira, L. A.; Rogers, L. B.; Gass, L. P.; Martin, G. W.; Irwin, R. M.; Von Wandruszka, R.; Berkowitz, D. A. Chem. Biomed. Envron. Instrum. 1980, 70, 249-271. (8) Sepaniak, M. J.; Yeung, E. S. Anal. Chem. 1977, 4 9 , 1554-1556. (9) McClain, W. M. Acc. Chem. Res. 1974, 7 , 199-205. (10) Kasha, M. Faraday SOC.Discuss. 1850, 9 , 14-19. (11) Lln, H. 6.; Topp, M. R. Chem. Phys. Left. 1977, 4 8 , 251-255. (12) Sepaniak, M. J. Ph.D. Dissertation, Iowa State University, 1960. (13) Sepaniak, M. J.; Yeung, E. S. J . Chromatogr. 1981, 277, 95-102. (14) Lin, H. B.; Topp, M. R. Chem. Phys. Lett. 1877, 4 7 , 442-447. (15) Novotny, M. Anal. Chem. 1881, 53, 1294 A-1308 A. (16) Knox, J.; Gilbert, M. J . Chromatogr. 1979, 786, 405-418. (17) Swofford, R. L.; McCiain, W. M. Chem. Phys. Left. 1975. 3 4 , 455-460.
RECEIVED for review December 14,1981. Accepted February 18, 1982. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research.
Agglomerated Pellicular Anion-Exchange Columns for Ion Chromatography Timothy S. Stevens” and Martln A. Langhorst Dow Chemical U.S.A., Michigan Division Analytical Laboratories, Midland, Michigan 48640
Slnce the conceptlon of ion chromatography, the bask technology for the anaiytlcal column packlng used for anion analysis has not changed. Mlcropartlcles of anlon exchanger (1000 to 50 000 A diameter) were agglomerated onto macropartlcles of cation exchanger (5-100 pm dlameter) to produce a low capacity pelilcuiar anion exchanger. The theoretical plate count for such columns Is low relative to modern practice, typlcaliy belng about 650. The use of smaller mlcropartlcles of anion exchanger (200-1 000 A dlameter) agglomerated onto efficlently packed beds of 15 pm cation exchanger resulted In Improved column performance wlth theoretlcai plate counts of about 2000. Separations that required 20 mln before, can now be obtalned In 10 min wlth better resolution.
Since the conception of ion chromatography the basic technology for the analytical column packing used for anion analysis has not changed. Microparticles of anion exchanger (1000 to 50 000 A diameter) were agglomerated onto macroparticles of cation exchanger (5-100 pm diameter) to produce
a low capacity “pellicular”anion exchanger (I). Improvements with this technology came with the use of monodisperse anion-exchange latex (2) rather than the previously used ground anion-exchange resin and by performing the agglomeration step in a polyvalent salt solution (3). The use of the monodisperse anion-exchange latex eliminated the need to refine the ground resin to obtain the optimum size range, and agglomerating in a polyvalent salt solution resulted in reproducible and dense agglomeration of the microparticles due to the resulting suppression of electrostatic repulsion forces between the microparticles. The performance of this type of exchanger is shown in Figure 1with a base line separation of fluoride, chloride, nitrite, phosphate, bromide, nitrate, and sulfate in 20 min using an eluent of 0.0024 M Na2C03,0.003 M NaHCO, at 138 mL/h with a 2.8 X 500 mm glass column filled with a packing composed of 3760 A diameter type 2 X 5 latex agglomerated onto 50 pm diameter surface sulfonated styrene divinylbenzene copolymer. Figure 1 is a reproduction of Figure 3 of reference 3. The theoretical plate count for the bromide ion peak in Figure 1is about 650. Theoretical place counts of about 650 for a 0.5 m long column are low by current liquid chroma-
0003-2700/82/0354-0950$01.25/00 1982 American Chemical Society
