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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. I n f . 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
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Anal. Chem. 1983, 55, 2451-2453
56
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.
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Figure 2. Response curves for phosphate. Details in text. Reagent ReI e IY 0 I,
Figure 1. Schematic of nanocolorlmeter. Details in text.
metal chelator. The phosphate reagent was a combination of ammonium paramolybdate and a catalyst in sulfuric acid added to a reducing reagent containing sodium acetate trihydrate, acetic acid, sodium metabisulfite, p-methylammoniumphenol sulfate, and a surfactant. Test solutions were placed in microcaps filled with paraffin oil which had been equilibrated with water and 100% C02 This was done to prevent the loss of calcium, magnesium, and phosphate (3). Pipettes and capillaries used in the analysis were siliconized with Prosil-28 (PCR, Gainesville, FL). An 8-10 nL pipette was used to transfer the test solution from the capillaries to the injection port of the colorimeter. The outside of the pipette. was rinsed with chloroform and deionized water. Before the transfer of test solution, the pipette wm placed in a holder which was affixed to a Brinkman micromanipulator (Brinkman Instruments, Toronto, Ontario). The transfer procedure was carried out under a stereomicroscope with a 30X magnification.
RESULTS AND DISCUSSION When calcium, magnesium, or phosphate was deleted from artificial tubule fluid, there was no significant effect from the other tubule fluid components. Calcium and phosphate were analyzed a t a flow rate of 20 nL/s with a 630-nm filter. Magnesium was analyzed at 18 n L / s with a 530-nm filter. The curves that were generated on the colorimeter were analyzed by weighing them on a Mettler balance (Mettler Instrument Corp., Hightstown, NJ). In order to maximize the area of the curves, various flow rates were tested. Variations in light intensity were also tested until the optimum combination of light and flow rate was established. Figure 2 demonstrates the sensitivity of the nanocolorimeter. These curves were produced by injecting 10 nL volumes of phosphate standard, ranging in concentration from 1.0 to 4.0 mM/L, into the cuvette. Calibration curves were generated by injecting 10-nL aliquots of calcium, magnesium, and phosphate into the flow-through cuvette. Each point on these curves was the mean of five measurements. The straight line plots were
obtained by linear regression analysis. The correlation coefficient for each line was 0.99. The introduction of bubbles into the reagent stream has been a major problem with this system. The cause of the problem has been the negative pressure produced by the withdrawal pump on tubing connections in the system. Bubbles produce noise peaks and interrupt the reagent flow. Maintaining a constant flow rate is essential for the accurate measurement of standard solutions and tubule fluid samples. By fabricating the injection port in the right arm of the cuvette and placing the right side of the cuvette directly into the reagent reservoir, we have achieved several benefits. Three tubing connections were eliminated tubing to cuvette, cuvette to injection port, and injection port to reagent reservoir. These were primary sites of bubble formation. The distance from the injection port to the end of the cuvette light path has been reduced to approximately 1.2 cm. This represents a substantial decrease in the dilution of the analyte as its development proceeded through the flow-through system. The change from a light emitting diode to a fiber optic system provided a significant increase in the sensitivity of the nanocolorimeter. This modification also gives the operator the flexibility of ion measurements at wavelengths for which there are no light emitting diodes. The fiber optic light sowce, along with the bubble-free flow-through cuvette, represents a significant improvement in the accuracy, sensitivity, and the ease of operation of the nanocolorimeter. Registry No. Calcium, 7440-70-2; magnesium, 7439-95-4.
LITERATURE CITED (1) Vurek, 0. G. Anal. Blochem. 1981, 114, 288-293. (2) Vurek, 0. Q.; Knepper, M. A. Kidney h f . 1982, 21, 656-658. (3) Muhlert, M.; Julita, M.; Quamme, G. Am. J . Physiol. 1982, 242, F202-F206.
RECEIVED for review June 17,1983. Accepted August 8,1983. This work was supported by National Institutes of Health Grant AH649.
Behavior of Trace Refractory Mlnerals In the Lithlum Metaborate Fusion-Acid Dlssolution Procedure Cyrus Feldman Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 In 1966, Suhr and Ingamells (1) suggested lithium metaborate (LiB02)as a fusion medium for most siliceous rocks. This salt, like other lithium borates, can be used to prepare samples for X-ray fluorescence. The authors also showed that the LBOz melt can easily be converted to a stable acid solution of the components without precipitating silica by quenching and dissolving the melt in 3% "OB. This solution can then be used for determining any component, including silicon, by emission spectrometry ( I ) or any other technique. Other authors have used various other mineral acids for quenching 0003-2700/83/0355-2451$01.50/0
and dissolution. In order to retard or prevent the precipitation of silica in a Li~C03-H3B03-SrC03-Co304fusion of rock powders, Govindaraju ( 2 , 3 )used dilute citric acid. Neither author was concerned with determining trace constituents. An excellent discussion of the use of lithium borate fusion in rock analysis is included in a review by Abbey, Aslin, and Lachance (4). It was desired to use this technique to determine trace impurities in rocks and other siliceous materials, using small disposable graphite crucibles. Preliminary experiments were 0 1983 American Chemical Society
