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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
ANALYTICAL CHEMISTRY, VOL. 55, NO. 14,
DECEMBER 1983 2449
.I
I
250
I
I
285
320
WAVELENGTH (nm) Flgure 1. Zeroorder UV spectra: (-) P-butanone; (- -) (- -) 2-propanone.
-
I
I
250
I 320
285
WAVELENGTH (nm)
3-heptanone;
considerably sharpened in the second derivative spectrum. This apparent band sharpening increases with increasing derivative order. Fine details, barely visible in the zero-order spectrum, are resolved in derivative spectra. These improvements in resolution also increase with increasing derivative order. Fine structure already present in zera-order spectra is further enhanced in derivative spectra. It follows then that second derivative spectra will be more detailed and sharper when the zero-order spectra are sharper and show fine structure. Therefore, utilization of this method to distinguish aliphatic and alicyclic ketones would appear to be one of the least successful applications. Before further discussion it should be emphasized that different recording conditions can cause changes in band position and band shape. Differences in the derivative gain may result in different band shapes. This is particularly true at the higher gain settings. Small wavelength shifts may also occur from varying the gain setting. Wavelength scanning speed will affect both the spectral amplitude and the background noise of the derivative spectrum. Increased scanning speed improves the signal to noise ratio. However, too fast a scan speed may cause peak slewing and perhaps small wavelength shifts. Varying absorbance range settings also results in differing amplitudes. Changing band-pass settings may lead to changes in resolution and band shape. Better resolution is obtained with decreasing slit width. All instrumental parameters should be optimized. Thereafter, they should be kept constant, and standards and samples should be run under the same conditions to ensure reproducible results. When zero-order spectra of the ketones used in this study were run in cyclohexane, only cyclopentanone showed fine structure. Cyclopentanone also had the highest absorption maximum, 300 nm. The other zero-order absorption maxima are as follows: cyclohexanone, 290 nm; 2-octanone, 280 nm; 3-heptanone, 279 nm; 2-propanone and 2-butanone, 278 nm (Figures 1 and 2). Figures 3 and 4 illustrate the second derivative spectra of the ketones examined. With the exception of cyclopentanone and cyclohexanone, the spectra do not show particularly sharp maxima and minima. However, every spectrum shows a very
Flgure 2. Zero-order UV spectra: (-) pentanone; (- - -) 2-octanone.
cyclohexanone; (-
-
-)
cyclo-
B
u
~
o
2
-
0
2
-
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WAVELENGTH (nm)
Second derivative UV spectra: cycloheptanone; (C) 2-octanone. Flgure 3.
250
285
320 250
285
(A)
320
cyclohexanone; (6)
250
285
320
WAVELENGTH (nm)
Figure 4. Second derlvatlve UV spectra: tanone; (C) 2-propanone.
(A)
2-butanone; (6)3-hep-
significant enhancement in spectral detail over the zero-order spectrum. The second derivative spectra of the ketones, for the most part, are markedly different, the maxima and minima occurring at different wavelengths. The only similarity appears between 2-octanone and 2-butanone. Even here the spectra can be distinguished by observing the obvious differences in the amplitudes of the maxima and minima; the 2-butanone spectrum shows noticeably larger amplitudes. Maximum to minimum measurements were made for five maxima and minima in the 2-octanone and 2-butanone spectra. The measurements were made for the following maxima-minima: 301-297,292-288,283-280,277-273,268-266 nm. The order of these amplitudes for 2-butanone is as follows: 301-297 > 292-288 > 277-273 > 268-266 > 283-280. The order for 2-octanone is 301-297, 292-288 > 283-280 > 268-266 > 277-273. The orders of the amplitudes are sufficiently different to distinguish between the two ketones. Spectra run at other concentrations proved that these amplitude orders remained the same at both lower and higher concentrations. Furthermore, differences in the shapes of the peaks are more subtle but, nevertheless, present. These results illustrate the value of second derivative spectra in the identification of ketones and emphasize the
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Anal. Chem. 1983, 55,2450-2451
versatility of this technique in general. In this case, the method has proven to be successful in differentiating compounds which have low intensity zero-order UV spectra with minimal or no fine structure. Furthermore, identification is possible with low concentrations (5 mg/L) without the need for derivatization. Registry No. 2-Propanone, 67-64-1; 2-butanone, 78-93-3; cyclohexanone,108-94-1;cyclopentanone, 120-92-3;3-heptanone, 106-35-4;2-octanone, 111-13-7.
LITERATURE CITED (1) Shriner, Ralph L.; Fuson, Reynold C.; Curtin, David Y.; Morrill, Terence C. "The Systematic Identification of Organic Compounds", 6th ed.; Wiley: New York, 1980; Chapter 6.
(2) Alpert, Nelson L.; Keiser, William E.; Szymanski, Herman A. "IR Theory and Practice of Infrared Spectroscopy", 2nd ed.; Plenum Press: New York, 1970; Chapter 5. (3) Silverstein, Robert M.; Bassler, G. Clayton; Morrill, Terence C. "Spectrometric Identification of Organic Compounds", 4th ed.; Wiley: New York, 1981; Chapter 6. (4) Gillam, A. E.; Stern, E. S. "An Introduction to Electronic Absorption Spectroscopy in Organic Chemlstry", 2nd ed.; Edward Arnold Ltd.: London, 1957; Chapter 5. (5) Hammond, V. J.; Price, W. C. J. Opt. Soc. Am. 1953, 43, 924. (6) O'Haver, T. C.; Green, 0. L. Inf. Lab. 1975, 5 , 11. (7) O'Haver, T. C.; Green, G. L. Anal. Chem. 1976, 48, 312. (8) Talsky, G.; Mayering, H.; Kreutzer, H. Angew. Chem. 1978, 90, 840. (9) Fell, A. F.; Proc. Anal. Div. Chem. Soc. 1978, 75,260. (IO) Lawrence, A. H.; MacNeil, J. D. Anal. Chem. 1982, 54, 2385. (11) Gill, R.; &I, T. S.; Moffat, A. C. J., Forensic Sci. Soc. 1982, 22, 165. (12) Davidson, A. G.; Elsheikh, H. Analyst (London) 1982, 707, 879.
RECEIVED for June 28,1983. Accepted September 12,1983.
Modlfied Flow-Through Colorimeter for Determination of Picomole Quantities of Calcium, Magnesium, and Phosphate J. Thomas Adkinson* and James C. Evans
Departments of Medicine and Physiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514 This paper describes the modifications made to a flowthrough nanocolorimeter, which is used for the measurement of picomole amounts of calcium, magnesium, and phosphate in aqueous solutions. The construction of this instrument was described by Vurek ( I ) . The modified nanocolorimeter has a quartz cuvette with a working volume of 115 nL and a light path of 0.5 cm. The injection port is approximately 7.0 mm from the cuvette light path. Nanoliter volumes of sample are injected into a reagent stream. The change in light absorption is quantitatively related to the concentration of analyte injected. A Gilmont micrometer syringe (Gilmont Instruments, Inc., Great Neck, NY) is linked to a Hampel perfusion pump (which has been converted to a withdrawal pump) to pull reagent through the cuvette (W. Hampel, Berlin, West Germany). Bubbles in the reagent stream have been eliminated. This was accomplished by elongating the injection side of the cuvette, forming a 90° bend in the quartz tubing, and placing one end in a reagent reservoir. A fiber optic light source, a photodiode, and an amplifier are used for colorimetric measurement. EXPERIMENTAL SECTION Apparatus. One piece of quartz tubing was used to fabricate the cuvette and the injection port. The quartz tubing has & outside diameter of 0.30 mm and an inside diameter of 0.25 mm (Friedrich and Dimmock, Millville,hrJ). A methane/oxygen torch was used to make the 90" bends and the windows that allow light to pass through the cuvette. This procedure was carried out as previously described by Vurek (2). After the windows were fornied in the two bends of the cuvette, the injection port was fabricated. The injection end of the cuvette was sealed with a CH4/O2torch. A piece of silastic tubing was connected to the opposite side of the cuvette. The tubing wak attached to a syringe and heat was judiciously applied to the injection site. Intraluminal pressure was increased and an opening was formed in the quartz tubing. This opening was reduceq in size to approximately 15 pm diameter by applying heat to the injection port. A 90" bend was formed approximately 5.0 mm distal to the injection port. The cuvette was placed on a metal block and secured in place with a silicone rubber adhesive. Exposed walls of the cuvette were covered with lamp black in an oil base in order to trap any light entering the walls of the cuvette. The cuvette-metal block was
attached to an X-Y manipulator. This allowed for the optimum positioning of the cuvette between the light source and the photodiode. Injection pipettes were made from the same quartz capillary as the cuvette. The pipette tips were drawn out to a fine point. The pipette was sealed into a 20-pL capillary (Microcaps, Drummond) which was used as the pipette holder. The volume of the pipette was measured by injecting aliquots of solution into an oil-filled 2-pL microcap. The microcap was calibrated by use of 3H-inulin. The colorimeter is contained ip an aluminum box 1.5 X 4.5 X 8.0 in. Figure 1shows its schematic. It contains a Fibrox Light source (Rank TayJor Hobson, Leicester, England) with a 150-W bulb. The intensity of the light can be changed by using an attenuator on the light source. A fiber optic likht guide 18 in. long and 2.0 mm 0.d. transmits the light from the source to the cuvette. The ac voltage of the Fibrox light source was rectified and filtered in order to convert it to dc voltage across the bulb filament. The {jght sensor is a general purpose photodetector transistor FPT-100. It is used as a photodiode; consequently, the emitter lead is not connected. The amplifier is made up of an integrated circuit operational amplifier 741 (National Semiconductor)used in the single-ended mode of input. It has an offset adjustment (offset voltage is summed with the photodetector current) and a gain adjustment range of 1to 20. The signal from the amplifier is received by an analog recorder. Reagents. Reagents used in the various tests were obtained from commercial sources. Artificial tubule fluid was used to check for any interference from substances present in normal kidney tubule fluid. The solution was made from reagent grade chemicals and deionized water. The artificial tubule fluid consisted of 140 mM NaCl, 1.5 mM MgS04,1.0 mM NaH2P04,2.6 mM CaCl,, 5.0 mM KC1, 25 mM NaHC03, 4.3 g/L urea, and 2.8 g/L D-glucose. Depending on the test performed, the appropriate ions were added or deleted. The reagents used for the analysis of calcium,magnesium,and phosphate p e available in prepared form (Pierce Chemical Co., RoCkford, IL). The calcium reagent consists of methylthymol blue and 8-qhinolinol, which is the magnesium complexor, a polyelectrolyte,and a monoethanolamine-sodium sulfite buffer. The magnesium reagent contains a calcium chelator, EGTA ([ethylenebis(oxyethylenenitrilo)]tetraacetic acid), and a dye reagent Calmagite (3-hydroxy-4-[(2-hydroxy-5-methylphenyl)azo]-1-naphthalenesulfonic acid) combined with KCN, a heavy
0003-2700/83/0355-2450$01.50/00 1983 American Chemlcal Society
