2445
Anal, Chem. 1983, 55,2445-2448
"1
3
LL
nm, 193.7 nm, and 197.2 nm. However, detection limits for arsenic were poor, about 100 pg/mL. Hence calibration curves and temperature effects were not investigated for arsenic. TGLs appear to be easier to use than microwave excited EDLs because the current method of wing hot air temperature stabilization for EDLs is more cumbersome than the heated filament used in TGLs. The high stability and excellent sensitivity obtained for AAS (2) and the good detection limits obtained here for dispersive AFS and elsewhere for nondispersive AFS (3) indicate that TGLs are a promising light source for atomic spectrometry. We plan to take a closer look a t the relative intensities of TGLs and EDLs by calibration of our monochromator with a calibrated deuterium arc in order to find out why we could not get better detection limits for arsenic.
P
j 0 ,
100
I
I
I
I
I
I
I
110 120 130 1LO 150 160 170 TGL OPERATING TEMPERATURE P C )
Figure 1. Effect of variation of operating temperature of two TGLs. The selenium concentration used was 100 pglmL. The cadmium concentration was 50 pg/L.
possible to explore the existence of the plateau normally seen for variation in operating temperature of EDLs (9). A plateau is generally thought to improve stability of EDLs (9) and probably improves warmup times of EDLs by making attainment of a particular temperature less critical. This could explain the longer warmup times noted here for TGLs operated at higher than the recommended temperature but it may be that the TGL power supply is slow to drive the temperature to the higher level. Calibration curves for cadmium and selenium were linear from the detection limit to about 4 orders of magnitude above the detection limit. This is as expected for AFS with intense line sources. Attempts were also made to obtain atomic fluorescence signals for three different arsenic TGLs at three lines, 235.0
ACKNOWLEDGMENT We acknowledge with thanks SGE, Inc., for providing the TGLs and their power supply. Registry No. Cadmium, 7440-43-9; selenium, 7782-49-2. LITERATURE CITED (1) Gough, D. S.; Sullivan, J. V. Anal. Chlm. Acta 1979, 108, 347-350. (2) Gough, D. S.; Sullivan, J. V. Anal. Chlm. Acta 1981, 124, 259-266. (3) Norrls, T.; Sullivan, J. V. Am. Lab. (Falrfldd, Conn.) 1982, 14 (12), 67-7 1. (4) Mlchel, R. G.: Coleman, Julia; Wlnefordner, J. D. Spectrochim. Acta, Parl B 1978, 338, 195-215. ( 5 ) Mlchel R. G.; Ottaway, J. M.; Sneddon, J; Fell, G. S. Analyst (London) 1878. - - - - ,103. . _ _ .1204-1209. . (6) Mlchel, R. G.; Ottaway, J. M.; Sneddon, J; Fell, G. S. Analyst (London) 1879. ... - , 104. . - , 687-691. . .. . (7) Seltzer, M. D.; Mlchel, R. G. Anal. Chem. 1983, 55, 1817-1819. (8) Browner, R. F.; Patel, B. M.; Glenn, T. H.; Rletta, M. E.; Wlnefordner, J. D., Spectrosc. Left. 1972, 5 , 311-318. (9) Browner, R. F.; Wlnefordner, J. D. Spectrochlm. Acta., Part B 1973, 288, 283-288.
RECEIVED for review July 18, 1983.
Accepted September 1, 1983. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for the partial support of this research.
Sample Deoxygenation for Fluorescence Spectrometry by Chemical Scavenging M. E. Rollie, C.-N. Ho, and I. M. Warner* Department of Chemistry, Emory University, Atlanta, Georgia 30322 Many organic compounds fluoresce and this property is widely used for analysis (1-3). The ground state of most organic molecules is a singlet state (i.e., all of the electrons in the molecule are spin paired). To produce measurable fluorescence emission, molecules must initially be promoted from the ground singlet state (designated So) to an excited singlet state. (The first and second excited singlet states are designated S1and S2,respectively.) This is commonly done by absorption of ultraviolet radiation. If the molecule is promoted to the second excited singlet state (SJ, it will rapidly undergo nonradiative deexcitation to the lowest vibrational level of the lowest excited singlet state (SI) by a combination of internal conversion and vibrational relaxation. Once the molecule reaches the lowest vibrational level of SI,it can return to So by emission of a photon. Thus, fluorescence is essentially the emission of a photon by an excited molecule that is returning to the ground state via a singlet-singlet transition (S, So). Generally, fluorescence emission occurs very rapidly
-
0003-2700/83/0355-2445$01.50/0
after excitation with lifetimes on the order of 1 X to 1 x 10-7 9. Various nonradiative deexcitation processes compete with and often greatly reduce fluorescence emission. Of these processes, quenching has the most pronounced effect. Quenching of fluorescence is defined as any process that results in a decrease in the true fluorescence efficiency of a molecule (4). Quenching processes divert the absorbed energy of the molecule into channels other than fluorescence (5). The presence of molecular oxygen contributes significantly to fluorescencequenching because most organic molecules in an excited state will nonradiatively deactivate after one or two collisions with molecular oxygen (6). Effects of oxygen quenching are most serious for solutions of aromatic hydrocarbons (7,81, but the fluorescence of virtually all organic compounds is quenched, at least slightly, by oxygen (9-12). Therefore, it is often important that samples be deoxygenated prior to fluorometric analysis. 0 1983 American Chemical Soclety
2446
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
__
__ ~
On+ 4Cr
Solution to be degassed
+ 4H
_
_
44 C r
~
_
t 2H,O
__ _.
Oxygen Seavinging Solution
cross section of solution undergoing deoxygenation through oxygen-permeable tubing. Flgure 1. A
Several methods of deoxygenation are currently used for preparation of fluorescent samples. These methods include nitrogen purging, freeze-thaw techniques, and preparation of samples within a vacuum. These methods have varying degrees of effectiveness. In addition, they are time-consuming and rather tedious. Thus, conventional sample deoxygenation is usually not done for routine fluorometric analysis despite obvious advantages. These problems have provided the incentive for developing a new approach to sample deoxygenation. This paper describes a novel method of sample deoxygenation that is based on the principles of oxygen diffusing across a permeable membrane in conjunction with the ability of some compounds to scavenge oxygen (12-14). Diffusion of oxygen across permeable tubing has been reported in the literature (13, 15). THEORY Our deoxygenation procedure is based on membrane permeability and the ability to generate a concentration gradient. In our system, the membrane is permeable to oxygen. The objective of the process is the minimization of the oxygen concentration in the fluorometric solution by way of oxygen diffusion from the solution, across the membrane, and into a scavenging solution in which oxygen is immediately consumed. Figure 1provides a diagram of the deoxygenation of a solution that is being pumped through tubing which is permeable to oxygen. The tubing is surrounded by a solution
N2 in
Figure 2.
of Cr2+ions which consumes the oxygen. Thus, as oxygen diffuses across the membrane to reduce the concentration difference, it is immediately consumed and reduced to water (Le., 0'-)in the presence of Cr2+and H+. The net result is that the concentration of oxygen never reaches a satisfactory level to reduce the concentration difference and, consequently, oxygen will continue to diffuse across the membrane until the analyte solution is oxygen free. This hypothesis assumes that migration through the solution and across the membrane is rapid and that the equilibrium of the scavenging reaction lies far enough to the right so that the amount of oxygen present in the scavenging solution at equilibrium is negligibly small.
Diagram of deoxygenation apparatus.
EXPERIMENTAL SECTION Deoxygenation Apparatus. The deoxygenation apparatus was developed by coupling a Gilson Minipuls 2 peristaltic pump and a Perkin-Elmer LS-5 fluorescence spectrophotometer, as is depicted in Figure 2. A solution of the analyte (1X lo-' M pyrene for all preliminary analysis) is pumped through oxygen-permeable tubing which is stored in a strongly reducing chromous sulfate, amalgamated zinc solution (12, 13). Oxygen diffuses from the analyte solution, through the tubing, and is reduced to water according to the following reactions:
-
Zn(Hg) + 2Cr3+
Zn2++ 2Cr2++ Hg
-
4Cr2++ O2 + 4H30+
4Cr3++ 6H20
(1) (2)
Static Experiments. Polypropylenetubing (2.76 mm internal diameter, 3.17 mm external diameter) containing 1 X lo-' M pyrene (Chem Service) in cyclohexane (Burdick and Jackson) was placed in a container of the chromous sulfate, amalgamated zinc reducing solution, The container was then placed in an ice bath to retard volatilization of the cyclohexane during the course of the experiment. The emission spectra of the deoxygenated pyrene solutions were compared to the emission spectra of pyrene solutions exposed to air. Enhancement factors of the deoxygenated pyrene solutions were calculated based on the luminescence intensity of several select peaks of the deoxygenated pyrene solutions relative to the intensity of the same peaks of the exposed pyrene solutions. Isochronal Experiments. The 1 X lo-' M pyrene (Chem Service) in cyclohexane (Burdick and Jackson) was allowed to equilibrate by shutting off the pump for selected increments of time. The emission spectrum of the analyte from 350 nm to 500 nm, at an excitation wavelength of 336 nm, was scanned at various
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14, DECEMBER 1983
2447
Table I type of tubing Teflon
a
length, ft
i.d./o.d., mm
12
0.8111.62
10
1.0211.78
10
silicone polypropylene
10
1.5913.18 2.7613.17
polyethylene
10
1.5712.08
equilibration time
enhancement re1 permeability factor dataa
none 2h 4h l h 2h 3h 3h none 15 min l h none 15 min 30 min 45 min l h
1.92 3.08 3.70 1.59 1.25 1.52 1.32
3.4 1.0 30.0
8.68 8.17
-1.7
2.21 3.34 6.74 10.41 12.60 13.40
-3.4
Manufacturer’s specifications normalized to Tygon.
time intervals during the equilibrium period to monitor luminescence enhancement as a function of time. Enhancement factors of the deoxygenated pyrene solutions were calculated as stated previously.
RESULTS AND DISCUSSION In order to determine the type of tubing (Le., opygen permeable membrane) that has the greatest permeability to oxygen, and therefore, the most rapid diffusion rate within the shortest analysis time, five types of tubing were evaluated. The results for the tubing available to us are shown in Table I. On the basis of these preliminary results polyethylene tubing has the most rapid oxygen diffusion rate within the shortest equilibration time and will therefore be the tubing used in subsequent experiments. Also included in Table I is a compilation of values from several manufacturers fo,r the permeability of the tubing to oxygen. The larger numbers depict greater relative permeability to oxygen. Despite the higher permeability value quoted for silicone tubing, it proved unsatisfactory for use in a flow system because of its tendency to expand under the pressure of the flowing solvent. In addition, relative differences in manufacturer’s relative permeability data and experimentally measured relative permeability can probably be attributed to differences in tubing wall thickness. We first performed a set of static experiments to evaluate the feasibility of our approach. The static experiments were performed in order to eliminate many of the variables that would be encountered in the isochronal flow system (i.e., flow rate, tubing length, tubing diameter, etc.). The static experiments were designed to test the validity of the underlying principles of the deoxygenation procedure (Le., to determine whether removal of oxygen by generation of a concentration difference across an oxygen permeable membrane was a plausible alternative to current methods of sample degassing). The graphical representation of the effect of equilibration time on enhancement factor for the static experiments is shown in Figure 3. The decrease in fluorescence observed for the second point in this curve has been observed ia several repeated experiments. The cause of this decrease has not been definitively determined. However, one explanation could be that quenching impurities may initially be leached from the tubing into the cyclohexane solution of the fluorophore. This causes an initial decrease in fluorescence intensity. When the impurities have reached an equilibrium concentration, the fluorescence no longer decreases but begins to increase as a result of oxygen diffusing through the tubing and being scavenged by the chromous solution. In spite of the initial ir-
E
7 t
!i
‘I-
/
r’
O m - m w m mm ~m m m m mb mm mm m mr nw mm m - wr mm mw m~ mm mm mm - - p .
TIME
(MINUTES)
Figure 3. Effect of sample equilibration time on deoxygenation procedure (static experiment).
CL
c
U LL
c W 2.
z I W
TIME (MINUTES)
Flgure 4. Effect of sample equilibration on deoxygenation procedure
(isochronal flow experiment).
regularity in the static curve, the overall objective of the static experiments was attained because the results showed conclusively that diffusion through an oxygen permeable membrane as the result of a concentration difference in conjunction with consumption of the oxygen by a scavenging solution is a simple and effective approach to sample degassing. It is evident from the enhancement factors that diffusion of oxygen
2448
Anal. Chem. 1983, 55,2448-2450
from the analyte solution has indeed taken place. Therefore, removal of oxygen from luminescent solutions via a concentration difference offers a viable alternative to current deoxygenation procedures. Figure 4 provides a graphical representation of the effect of equilibrium time on enhancement factor for the isochronal experiments. The greatest luminescence enhancement appears to occur a t 90 min equilibration time.
in equilibration time can be obtained by using tubing of decreased wall thickness. Finally, it is apparent to us that this approach will be most useful when incorporated into a flow injection analysis (FIA) system. Such studies are currently under way. Registry No. Oxygen, 7782-44-1; chromium, 1440-47-3.
CONCLUSIONS Although ow work is somewhat preliminary, we believe that chemical deoxygenation has great potential as a simple and effective alternative to current methods of sample degassing. In addition, since most fluorophores have much shorter lifetimes than pyrene and are, therefore, less susceptible to oxygen quenching, we recognize that the greatest utility may be derived from the use of chemical deoxygenation for the preparation of phosphorescent samples. One of the major problems with room temperature phosphorescence in liquid solution is that the samples require rigorous degassing in order to alleviate oxygen quenching (16,17). Chemical deoxygenation provides a simple and effective method of phosphorescence sample preparation and, therefore, offers a superior alternative to the present techniques. Further work is in progress to optimize our degassing procedure by monitoring the amount of oxygen directly and to utilize the technique for practical analytical applications. We believe that an organic scavenging agent may be more effective for degassing the solutions since such a solvent will provide better wetability of the tubing than will an aqueous solvent. In addition, considerable reduction
O’Donnell, C. M.; Solie, T. N. Anal. Chem. 1978, 50, 189R-205R. Wehry, E. L. Anal. Chem. 1980,52, 75R-90R. Wehry, E. L. Anal. Chem. 1982,54, 131R-148R. Parker, C. A.; Rees, W. T. Analyst (London) 1962,87, 83-1 11. (5) Parker, C. A. “Photoluminescence of Solutions with Applicatlons to Photochemlstry and Armlytlcal Chemlstry”; American Elsevier: New York, 1968. (6) Parker, C. A.; Rees, W. T. Analyst (London) 1980,85, 587-600. (7) Smith, 0.J. Chem. Soc., Faraday Trans. 1982, 78. 769-773. (8) Darmanyan, A. P. Chem. Phys. Lett. 1982,86, 405-410. (9) Vidaver, W.; Popovlc, R.; Bruce, D.; Colbow, K. fhotochem. fhotob i d . 1981,34, 633-636. (10) Whltaker. T. J.; Bushaw. B. A. J. fhys. Chem. 1981,85, 2180-2182. (11) BumJarner,S. L.; Schuh, M. D.; Thomas, M. P. J. Phys. Chem. 1982, 86, 4029-4033. (12) Kolthoff, I . M.; Lingane, J. J. ”Polarography”; 2nd ed.; Interscience: New York, 1952, Vol. I. pp 396. (13) Authur, P. Anal. Chem. IS64, 3 6 , 701-702. (14) Freeman, T. M.; Seltz, W. R. Anal. Chem. lS81, 53,98-102. (15) ERG3110 Degasser, Manufacturer’s Literature, Product of Erma Optlcal Works, Ltd., 2-4-5 Kajlcho, Chiyoda-Ku, Tokyo, 101 Japan. (18) Miller, J. N. Trends Anal. Chem. lS81, 1 , 31-34. (17) Cline Love, L. J.; Skrilec, M.; Habarta, J. G. Anal. Chem. 1980,52, 754-759.
LITERATURE CITED (1) (2) (3) (4)
RECEIVED for review June 7,1983. Accepted August 23,1983.
US.and international patents are pending on the method described.
Identification of Ketones by Second Derivatlve Ultraviolet Spectrometry Larie Meal Chemical Technology Department, University of Cincinnati, Cincinnati, Ohio 45210 Several common procedures are employed in the characterization and the identification of ketones. Among the wet techniques are derivative preparations of oximes, 2,4-dinitrophenylhydrazones, and semicarbazones (1). Infrared techniques have long been used as an instrumental method for this identification and characterization work (2). Ultraviolet (W)spectra are valuable in the characterization of ketones because of the typical, low-intensity, often featureless bands that occur in the 270-300 nm range (3). However, the low intensity and the lack of fine structure render the method less useful for identification. In addition, many ketones have absorption maxima at or near the same wavelengths. UV spectra of the semicarbazones and 2,4-dinitrophenylhydrazones aid in identification mainly because of the much larger absorptivities of these derivatives. While this derivatization produces some wavelength differences in absorption maxima, many still have very similar absorption characteristics ( 4 ) . The application of derivative spectrometry to UV spectrometry was first described in 1953 (5). Later, information was presented that discussed the application of the technique to hypothetical situations and possible systematic and random errors (6, 7). Various methods for the generation of derivative spectra have been reviewed (8) and the modified derivative functions that result from obtaining a derivative spectrum by electronic means have been reported (9). Recently ultraviolet-visible derivative spectrophotometry has begun to see wider application as a valuable qualitative
method (10-12). This paper will describe the use of second derivative UV spectrophotometry in the identification of ketones. EXPERIMENTAL SECTION Reagents. 2-Propanone (MCB), 2-butanone (Baker), and cyclohexanone (Mallinckrodt), all reagent grade, and cyclopentanone, 3-heptanone)and 2-octanone (all Eastman, practical grade) were used without further purification. Apparatus. Spectral data were recorded with a Pye-Unicam, Model 8-100,recording UV-VIS spectrophotometer with first and second derivative accessory. Quartz cells of 1-cm path length were used. The spectra were obtained with the following instrumental parameters: bandwidth, 1nm; wavelength speed, 1nm/s; chart speed, 5 s/cm; absorbance, 2. The derivative accessory was set for second derivative measurement at gain 3, the highest undamped setting. Procedure. All scans were made in cyclohexane (Eastman, Spectro ACS) solution. The ultraviolet region was scanned from 320 to 250 nm in all cases. Concentrationsof the ketones were approximately 5 mg/L. RESULTS AND DISCUSSION The second derivative ultraviolet spectrum could be defined as the rate of change of gradient (d2A/dX2) plotted VS. wavelength (A). The zero-order absorption maximum appears as a minimum and the points of inflection (zero-ordercurve) appear as maxima. The second derivative spectrum has a central minimum with a maximum symmetrically positioned on either side. Therefore, the original (zero order) peak is
0003-2700/83/0355-2448$01.50/00 1983 American Chemical Society
