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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
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for 1h. Conversion to 11s form was nearly 100% as monitored
by isokinetic sucrose gradient (3)sedimentation. 'H FT NMR studies (in DzO)of the flexibilities of a variety of bound inhibitors of this enzyme are reported elsewhere ( 4 ) . For the experiment shown in Figure 1, tritium activity was measured with a liquid scintillation counter (Nuclear Chicago, Mark V, Chicago, IL), and AchE activity was determined by the rate of hydrolysis of acetylthiocholine ( 5 ) . The sedimentation proceeded for ca. 20 h at 40000 rpm in a Beckman 23-50 ultracentrifuge. LITERATURE CITED (1) Lange, N. A., Ed. "Handbook of Chemistry", 10th ed.; McGraw-HIII: New York, 1961; pp 329, 1189.
511 - 4
tboitorn
10 20 F r a c t i o n Number
30 iop-
Figure 1. Concentration of acetylcholinesterase (AchE) into D20buffer. AchE in tritiited H20 buffer was layered on top of D20 buffer and the sample centrifuged at 40 000 rpm for several hours. AchE concentration (0)is then reflected by the activky assay scaled by the left-hand axis, and diffusbn of tritiated H20 outward in the centrifuge tube is reflected by the radioactlvity profile (A)scaled by the right-hand axis. Fractions are numbered starting from the bottom of the centrifuge tube.
C. 3.1.1.7) was isolated from fresh electroplax tissue by the affinity chromatography method of Webb and Clark (2) and converted to 11s form by incubation with trypsin 1 mg/25 mL; A2@= 0.7)
(2) Webb, G.; Clark, D. 278-288.
G. Arch. Blochem. Biophys. 1078, 797,
(3) Miller, D. B.; Christopher, J. P.; Borrough, D. Biophys. Chem. 1978, 9 , 9-14.
(4) (5)
Marshall, A. G.; Carruthers, J. M. Mol. Pharmacol., in press. Ellman, 0. L.; Courtney, K. D.;Andre$, V.; Featherstore, R. M.
Bio-
chem. Pharmacol. 1961, 7, 88-95.
RECEIVED for review January 12, 1981. Accepted March 9, 1981. This work was supported by grants (to A.G.M.) from Natural Sciences and Engineering Research Council of Canada (A-6178), University of British Columbia, U.S. Public Health Service (N.I.H. GM-29274-Ol), and the Alfred P. Sloan Foundation (Research Fellow, 1976-1980).
