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extraction. Because some transport of the uncomplexed ion will occur, separation factors will not be as great as by extraction; however, Donnan dialysis has some potential advantages. It can be adapted to continuous monitoring of streams (7), and the aqueous receiver system can be coupled to certain analytical methods more readily than the organic phase of solvent extraction. Presently the use of specific complexing agents is being explored to extend the range of application. LITERATURE C I T E D (1) Lundquist, G. L.; Washinger, G.; Cox, J. A. Anal. Chem. 1975, 4 7 , 319-322. (2) Blaedel, W. J.; Kissel, T. R. Anal. Chem. 1972, 44, 2109-2111. (3) Cox, J. A.; Cheng, K. H. Anal. Lett. 1978, 11, 653-660. (4) Cox, J. A.; Twardowski, 2 . Anal. Chim. Acta 1980, 119, 39-45. (5) Cox, J. A,; Twardowskl, 2 . Anal. Chem. 1980, 52, 1503-1505.
(6) Cox, J. A,; DiNunzio, J. E. Anal. Chem. 1977, 49, 1272-1275.
James A. Cox* E w a Olbrych Department of Chemistry and Biochemistry Southern Illinois University at Carbondale Carbondale, Illinois 62901 Krystyna B r a j t e r Department of Chemistry University of Warsaw Warsaw 02-093, Poland
RECEIVED for review February 17,1981. Accepted April 10, 1981. This work was supported by the National Science Foundation under Grant CHE-7908660.
AIDS FOR ANALYTICAL CHEMISTS Teflon FEP Coil as Reactor for the Photochemical-Conductivity Detection in Liquid Chromatography Paolo Clccioll, Rem0 lappa, and Alfred0 Guiducci Istltuto sull’lnauinamento Atmosferico del C.N.R., Area della Ricerca di Roma, Via Salaria Km 29.300, C.P. 10. 000 16 Monterotondo Scalo, Italy
In the last few years, many specific detectors have been developed for high-performance liquid chromatography (HPLC). Among them, photochemical-conductivity detector (PCD) (1,2)seems to possess some unique features which may make it particularly valuable for the solution of trace organic problems, especially in the environmental, biological, and pharmaceutical areas. These features can be summarized as follows: (1) high selectivity for the detection of halogenated as well as many nitrogen- and sulfur-containing compounds; (2) high sensitivity allowing the determination of several compounds in the nanogram or, in some instances, low picogram range; (3) simple design which makes the PCD inexpensive and largely accessible to any laboratory. However, the achievement of the optimal PCD working conditions presents some operational problems which render its use difficult and time-consuming. The reasons of these difficulties can be understood by analyzing in detail the PCD working mechanism. Basically the PCD is comprised of a splitting device, a postcolumn reactor, and two conductivity cells placed in parallel. The sample emerging from the column is split in two streams. On the analytical side of the detector, the sample passes through a quartz reaction coil where it is irradiated by an intense UV lamp functioning as ionizing source. This effluent then passes into the analytical conductivity cell. On the reference side, the sample passes through a Teflon delay coil having the same dimensions as the quartz reaction coil and then reaches the reference conductivity cell. The signal is obtained as an output of the difference in conductivity of the irradiated and nonirradiated flow paths of the sample. Optimal sensitivity and stable base line result when the amount, band spreading, and delay time of the samples passing through the analytical and the reference cell are the same. Since these parameters are difficult to control when different materials are employed for the construction of the UV reactor and the delay coil, the flow rates passing through the two cells need frequent adjustments. This
operation is very critical and time-consuming because no flow controllers connected to the cells outlet can be employed. As such devices generate an overpressure within the detector, the disconnection (or even the breakage) of the quartz reaction coil might occur. For the same reason no flow rates exceeding 3 mL/min can be employed with the PCD, and it must be placed as the last component when used as a part of a multidetection unit. Since the devices developed for reducing the formation of gaseous bubbles into the liquid stream cannot be employed, it is difficult to carry out analyses with volatile eluants (such as methanol) when flow rates lower than 0.5 ml/min are needed. The aim of the present work is to suggest a simple and inexpensive way to overcome the above limitations and to improve the operability and the versatility of the PCD without affecting its overall performances. EXPERIMENTAL SECTION A Tracor PCD Model 965 (Austin,TX) in tandem with a Varian 5000 liquid chromatgraph (Varian, Walnut Creek Division, CA), was used for our investigations. A Variscan UV absorbance detector was used as an additional component for the multidetection unit. Two different columns were employed for the chromatographic analyses: a 25 cm X 4 mm i.d. stainless steel column packed with Micropack MCH (10 pm) supplied by Varian and a homemade 25 cm X 2 mm i.d. glass column packed with Carbopack B (Supelco, PA) 25-33 pm (3). Teflon FEP tubing 1/16 in. 0.d. X 0.015 in. i.d.) was employed for the construction of the reaction coil. It was supplied by Tracor as a part of the accessory kit of the Tracor 965 PCD. RESULTS A N D DISCUSSION In a recent work, Frei et al. (4)have tested several materials for making postcolumn reactors for HPLC. According to these authors, Teflon TFE works better than quartz when UV-induced fluorescence is used as the detection method in liquid chromatography. Because significant advantages can be gained by avoiding the use of the quartz postcolumn reactor for the photochemical-conductivity detection in liquid chro-
0003-2700/81/0353-1309$01.25/0 0 1981 Amerlcan Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 53, NO. 8, JULY 1981
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Comparison between the photochemical-conductivity response obtained by using quartz and Teflon coils as reactor: (a) sample, Diuron; column, Micropak MCH 10 Fm (30 cm X 4.5 mm i.d.): eluant, CH30H-H20, 70:30; sensitivity, 10 X 20 X 20 mV full scale; flow rate, 1 mL/min; UV lamp, 254 nm; (b) sample, Atrazine; same experlmental conditions as (a); (c) sample, 2,3,7,8-TCDD; column, Micropak MCH 10 pm (30 cm X 4.5 mm i,d.); eluant, CH30H; sensitivity, 10 X 10 X 10 mV full scale; flow rate, 0.5 mL/min; UV lamp, 214 nm. Figure 2.
matography, the conventional PCD was modified by substituting the quartz reaction coil with Teflon FEP tubing having the same internal diameter and the same length. Teflon FEP
(a perfluorinated polymer having mechanical properties very close to Teflon TFE) was chosen because the delay coil as well as all the connecting lines of the Tracor 965 PCD were made with tubing of this material. In this way, the analytical side of the detector was made identical in volume and in material to the reference side. Because Teflon tubing is soft and easy to cut and connect with the metal body containing the conductivity cells (2),the modification can be accomplished in very short time without any difficulty. The schematic diagrams reported in Figure l show how the quartz and Teflon FEP reaction coils were positioned with respect to the UV lamp in the conventional design (a) and after our modifications (b). As can be seen, the Teflon FEP reaction coil was placed in contact with the aluminum body surrounding the UV lamp, consequently the distance between the reactor and the irradiating source was in our case longer than that originally employed in the Tracor 965 PCD. Although this solution certainly decreases the intensity of the UV radiation reaching the Teflon FEP reactor, it has been found easier to accomplish because it does not require the use of any special device for supporting the reaction coil. The PCD responses obtained with quartz and Teflon FEP reaction coils were compared by injecting known amounts of Diuron, Atrazine, and 2,3,7,8-
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Anal. Chem. 1981, 53, 1311-1312
tetrachlorodibenzodioxin into the analytical column. The photoionization of the former two compounds was accomplished with the mercury lamp (254 nm) whereas the latter compound was irradiated with the zinc lamp (214 nm). All the analyses were carried out under the same chromatographic conditions and the same procedure was followed for optimizing the PCD. Figure 2 shows an example of the results obtained. As can be seen, no major difference in the photochemical ionization efficiency exists between Teflon FEP and quartz within the experimental errors (10%). The slight decrease in sensitivity observed for Diuron and Atrazine with respect to the results shown by Popovich et al. (2) was attributed to the presence of small quantitites of contaminants contained in our water solutions. In any case, the presence of interfering compounds was systematically affecting both sets of measurements carried out with Teflon FEP and quartz. Although the use of Teflon FEP does not improve (at least in our case) the photochemical ionization efficiency, it certainly improves the operability and versatility of the PCD. Because Teflon FEP tubing can easily stand pressures of the order of 5 kg/cm2, metal capillaries can be connected to the cell outlets for eliminating base line drift. Actually, we found that a metal capillary (1 m x 0.25 mm id.) was sufficient to maintain the flow rates passing through the conductivity cells steady for more than 3 weeks. Flow rates ranging from 0.1 to 6 mL/min were also employed without any particular problem. On the basis of the previous results we have built a multidetection unit in which the PCD was placed as the first component. A UV absorbance detector was connected to the PCD outlet. Although this configuration is not the most advantageous because the PCD destroys a portion of the eluted sample, it
conveniently demonstrates the increased versatility of the PCD equipped with a Teflon FEP reaction coil. Figure 3 shows two examples of the operation of this unit. Figure 3a reports an example of chromatographic analysis in which the two detectors exhibit a relatively high response for the eluted compounds. The results of this figure not only indicate that the presence of the UV absorbance detector does not affect the PCD performances but also show that the band broadening occurring within the PCD does not significantly alter the efficiency and resolution of the chromatographic column. Figure 3b shows the analysis of a pesticide extract, where it has been possible to identify the presence of Endrin by comparing the different responses measured with the two detectors. During 6 months of continuous operation constant sensitivity coupled with satisfactory base line stability has been obtained with the multidetection unit described above. The use of this unit has been found particularly valuable for studying the photochemical decomposition of many chlorinated pollutants occurring in liquid solutions.
LITERATURE CITED (1) Rogers, D. H.;Hall, R. C. “Separations and Response Factors of Selected Pesticide Compounds by HPLC”; Paper presented at the Plttsburgh Conference of Analytical Chemistry, Paper No. 8, 1977. (2) Popovich, D. J.; Dixon, J. B.; Ehrlich, 6 . J. J. Chromatogr. Sci. 1979, 17, 643-650. (3) Ciccioli, P.; Tappa, R.; Di Corcla, A.; Llberti, A. J. Chromatogr. 1981, 206, 35-42. (4) Sholten, A. H. M. T.; Brinkman, U. A. Th.; Frei., R. W. J. Chromatogr. 1980, 191,239-248.
RECEIVED for review November 13, 1980. Accepted March 20, 1981.
Concentration of Aqueous Macromolecules into Deuterium Oxide by Ultracentrifugation Alan G. Marshall” and Junko M. Carruthers Deparfments of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 432 10
‘H nuclear magnetic resonance (NMR) experiments for aqueous macromolecules are typically conducted in deuterated water for two reasons. First, the DzO 2H NMR signal provides a convenient field-frequency “lock” to keep the magnetic field strength constant. Second, the huge ‘H NMR peak from pure HzO can lead to dynamic range problems and can also mask underlying small peaks from the (necessarily) dilute large molecule. Since biological macromolecules are normally isolated from an HzO medium, it is necessary to exchange the H 2 0 for D 2 0 for such NMR measurements. If the macromolecule is stable to lyophilization, then the aqueous sample may simply be freeze-dried and dissolved in DzO buffer one or more times to complete the exchange. Alternatively, the aqueous sample may be dialyzed against a large volume of DzO until the exchange is complete. Unfortunately, many macromolecules (in this case, electric eel 11s acetylcholinesterase, AchE, 330 000 dalton) are denatured by lyophilization. Moreover, ultrafiltration of this membrane-bound protein can result in large losses (35% loss of activity for AchE) when such hydrophobic molecules contact the. dialysis membrane. Another method for replacing H 2 0 by DzO is suggested by the higher specific gravity of DzO compared to H20 (1.108 vs. 0003-2700/8 1 /0353-13 1 1$01.25/0
0.997 a t 25 “C) ( I ) , as shown in Figure 1. A concentrated sample of 11sAchE in tritiated HzO was carefully layered on top of protein-free DzO buffer in a centrifuge tube. After several hours of high-speed centrifugation, aliquota withdrawn from the bottom of the tube showed the 3H and AchE activities plotted in the figure. It can be seen that most of the AchE has concentrated near the bottom of the tube, with negligible diffusion of HzO (made visible from 3H activity) to that region. In practice, this method is made difficult by convection currents due to nonuniform sample temperature during the run and by the need to calibrate the length of the run so as to stop the centrifuge a t the moment when the protein is well-concentrated near the bottom of the centrifuge tube but not yet pelleted against the bottom wall. Attempts to slow the sedimentation near the bottom by increasing the density with added salts or sucrose failed. Nevertheless, the method has been made to work successfully with AchE and should be considered when (as in this case) other techniques are not suitable.
EXPERIMENTAL SECTION Electric eel acetylcholinesterase (acetylcholine hydrolase, E. 0 1981 American Chemical Society
