ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
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(2 = 5 2 ) . Results are shown in part c of Figure 5.
CONCLUSIONS
(Cl
t
I
1
iJ
Atomic number
Figure 5. Intensity of the Raman band obtained by scattering of silver K radiation on K (a) and L electrons (b), and tin K radiation on L electrons (c), all relative to the total K, resp. L fluorescence as emerging from "infinitely" thick specimens
tative estimate for the intensity of the Raman band, obtained in the way described above, is represented in part b of Figure 2; the decreasing tendency with increasing atomic number is clearly apparent. In order to examine the presence of the effect for other excitation energies, the experiments described above were repeated using the silver secondary target and filter assembly. Because of the larger energy difference between coherently and incoherently scattered exciting radiation in this case, Raman scattering on the K shell was difficult to observe. Only for two elements, sulfur and chlorine, the effect could be detected conclusively. For scattering on L electrons, however, results comparable to those found for the molybdenum excitation were obtained for elements in the range rhodium (2 = 45) to tellurium (2= 52). Two examples are shown in Figure 4. From the quantitative estimates shown in parts a and b of Figure 5 , it can be seen that the results for molybdenum and silver K excitation are comparable in magnitude; the decrease in intensity for increasing atomic numbers is again directly apparent. Finally, tin K excitation was examined. Whereas no data could be obtained for scattering on K electrons, a similar behavior as the one described above was found for the L electrons of the elements from cadmium (2= 48) to tellurium
The results of the experiments show that Raman structures can emerge unexpectedly in X-ray spectra although the phenomenon was never reported before in analytical energy dispersive spectrometry. As an example, for routine analyses in our laboratory using molybdenum K excitation, a thin zirconium wire is always suspended below the sample to act as an external monitor for tube output and possible malso that the function of the dead time correction circuitry (3, Raman feature is present in all spectra. In this case, it can be easily corrected for, of course, since its intensity is always proportional to the intensity of the Zr K a peak. The effect can occur for a wide variety of samples and excitation conditions however, its sharp high energy edge giving it sometimes a peak-like appearance which might easily be confused with a normal fluorescence peak. Errors in background subtraction can also result, leading to erroneous determination of trace element concentrations. As a thumb rule, whenever mono- or bichromatic K radiation froin an element with atomic number Z is used for excitation, Raman bands can be expected to show u p in the spectra of samples consisting predominantly of elements with atomic number from Z - 2 to Z 7. Moreover, when the energy of the exciting radiation is between 15 and 25 keV, the phenomenon may occur for samples of elements with atomic number ranging from about 13 to 17. The use of thicker samples will enhance the observed intensity of the effect as compared to the fluorescence radiation of the element examined. We feel that every user of energy-dispersive X-ray spectrometry should be aware of the possible occurence of Raman bands in his spectra, and recognize the important consequences they may have for the accurate analysis of trace constituents.
+
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7)
Raman, C. V., Indian J . Phys. 1928, 2 , 387. Smekal, A,, Naturwissenschaften, 1923, 11, 873. Das Gupta, K. Phys. Rev. Lett. 1959, 3 , 38. Suzuki, T . J . Phys. Soc. Jpn. 1987, 2.2,1139. Sparks, C. J., Jr. Phys. Rev. Left. 1974, 33, 262. Bannett, Y. 6.; Freund, I. Phys. Rev. Lett. 1975, 3 4 , 372. Van Espen, P.; Adams, F . Anal. Chem. 1976, 48, 1823.
RECEIVED for review February 16,1979 Accepted April 2,1979.
Flow System for Sensitive and Reproducible Chemiluminescence Measurements H. R. Schroeder" and
P. 0. Volgelhut
Ames Research and Development Department, Division of Miles Laboratories, Inc., 1127 Myrtle Street, Elkhart, Indiana 46514
We have shown that chemiluminescence can be used to monitor competitive protein-binding reactions (1-3). Chemiluminescent reactions of aminophthalhydrazides with the H z 0 2(oxidant) and microperoxidase (catalyst) are relatively rapid ( 4 ) ; therefore, the reactants must be mixed quickly in front of the photodetector. Various flow systems have been described for reproducible measurement of chemiluminescence, b u t these systems utilize aminophthalhydrazide concentrations above micromolar (5-9). Since competitive protein-binding reactions employ aminophthalhydrazides at nanomolar and picomolar levels, we investigated methods for mixing reagents for reproducible measurements of chemiluminescence. Initially, a solution of 0003-2700/79/0351-1563$01 .OO/O
the oxidant was injected into a small test tube containing the aminophthalhydrazide and a catalyst; however, the reproducibility of the measurements was not completely satisfactory ( 4 ) . Here we describe a flow system which gives substantially improved reproducibility and retains the high sensitivity of previous systems.
EXPERIMENTAL Apparatus. A flow cell shown in Figure 1was machined from Lucite. The reaction chamber (8 ML)was formed by a cylindrical cavity with a conical bottom and a removable concentric plug with polished faces and sealed by an O-ring. The two reagent streams
were admitted into the cavity through opposing sapphire orifices (0.001-in. diameter, Swiss Jewel Co., Philadelphia, Pa.) to provide 0 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
‘ I = -
F n
5 -
- 50
WASTE
I
PM
%
i
Figure 1. Schematic drawing of a cross section of the RMF system. (A) Sapphire orifice, (B) reaction chamber, (C) polished Lucite plug (window),(D) plug, (E) flange fittings, (F) syringes and drive
jet action for fast mixing. The exit port was located axially at the apex of a shallow cone to facilitate removal of bubbles. The cross sectional area of the exit ports was equal to the sum of the areas of the entrance orifices. Two 5-mL disposable glass syringes were connected to the sapphire orifices through black Teflon tubing of 0.034-in. i.d. and 0.012-in. wall thickness (Fluoro Plastics, Inc., G and Venango St., Philadelphia, Pa. 19134) with 1/4-28 fittings from Altex (Berkeley, Calif.). Both syringes were driven simultaneously with a Multispeed Transmission Drive (Harvard Apparatus Co., Dover, Mass.). The cell was mounted directly in front of the photodetector of a DuPont 760 Luminescence Biometer with the sample chamber of the cylinder-rotor in the “expose” position. The injection port of the Biometer was removed and the hole beneath it enlarged slightly to accommodate the three Teflon tubes from the flow cell. This access port was made light-tight. Light Measurements. Rapid Mining Flaw (RMF)System. A reagent (5 mL) containing diethylisoluminol, 50 mM NaOH, 19 mM 5.5-diethvl barbiturate. and 2.5 uM microDeroxidase at a final pH of 12.6 were placed in one of the syring’es of the flow apparatus. The second syringe contained 5 mL of 90 mM H202 in 10 mM Tris-HC1, pH 7.4. Both syringes were driven simultaneously at flow rates of 1.36 mL/min/syringe (or other speed as indicated) to mix the reagents in the flow cell and produce chemiluminescence. Light intensity was recorded on a Hewlett-Packard recorder (Model 17501A) which was connected to the Biometer at a sensitivity setting of 900with 0-100 mV output. Single Injection ( S I )Sjstem. In an alternate method, similar chemiluminescent reactions were carried out by automatic injection ( 4 ) of H 2 0 2(10 FL) from 4 cm above the surface of 150 p L of reagent in 6 X 50 mm test tubes mounted in the Biometer. The sensitivity setting of the Biometer was 820 and reaction profiles were obtained with an attached custom-built microprocessor. Light Standard. Two hundred pL of aquasol (New England Nuclear, 549 Albany St., Boston, Mass.) scintillation fluid containing 0.5 (I) and 1.0 WCi (11) of I4C-phenylalanine was sealed in 6 X 50 mm test tubes. Disintegration of I4C caused emission of a continuous output of photons which served as a constant light source of similar intensity as that from chemiluminescent reactions. Solutions. Stock solutions ( 4 ) of diethylisoluminol (1mM) in 100 mM Na2C03,pH 10.5 and microperoxidase (200 FM) in 10 mM Tris-HC1, pH 7.4, were stored a t 4 “C. These solutions were diluted in 75 mM 5,j-diethyl barbiturate buffer, pH 8.6, prior to use. The water used to make up reagents was charcoal filtered and glass distilled.
RESULTS AND DISCUSSION A R M F system was designed to improve the precision of previously reported light measurements (4) for quantitative measurement of chemiluminescent compounds. Diethyl-
Figure 2. Chemiluminescencemeasurements with a RMF system and a SI system. Chemiluminescence from reactions containing a final level of 38.6 pM diethylisoluminol were conducted with the RMF system and monitored with the Biometer as described in the Experimental section under light measurements. Light production shown in a copy of the recorded trace (-) rapidly attained a steady state, but ceased when flow was stopped and resumed the earlier level with return of original flow rate. Ten 1 L of 10 mM H,O, was added with the automatic SI system ( 4 ) to produce light in reaction mixtures (150 kL) containing 25.7 pM diethylisoluminol,0.27 1 M microperoxidase, 57.5 mM 53diethylbarbiturateand 0.1 M NaOH at pH 12.6 in 6 X 50 mm test tubes (0-0). Chemiluminescence was monitored with the Biometer (sensitivity 660) and a microprocessor at a 10-ms sampling rate ( 4 )
-
I
m
0
I
0.1 0.2 0.3 0.4 0.5
1.0
Flow Rate (ml/min./Syringe)
Figure 3. Effect of flow rate on light intensity produced by oxidation of diethylisoluminol. Reactions were carried out with 38 pM diethylisoluminol and the RMF system as described in the Experimental section except that the flow rate was varied as indicated
isoluminol (anale),microperoxidase, and base were combined in one syringe and rapidly mixed in the flow cell with HzOz from a second syringe to produce light. To vary diethylisoluminol concentration, a new solution containing the desired level was placed in t h e first syringe and t h e operation was repeated, since the experimental apparatus lacked automatic sampling capability. Representative light production curves for both the RMF and SI system are shown in Figure 2. About 0.5 s after initiation of flow, steady state chemiluminescence was observed until the flow was interrupted. The intensity of light produced by 38 p M diethylisoluminol increased with flow rate and reached a maximum a t 0.68 mL/min/syringe (1.36 mL/min/total flow) (Figure 3). This rate was twice t h a t necessary t o displace the cell contents in the time required for peak light intensity (0.7 s), which was determined by kinetic measurements using the SI system (Figure 2). Thus, even a short 10-s measurement for example, is equivalent to averaging a multitude of light peaks produced in the SI system and increased reproducibility would be expected. Furthermore, the linear dose response and detection limit obtained with diethylisoluminol in the RMF system was similar to that with the highly sensitive SI system (Figure 41, thereby satisfying one of the major goals. T h e R M F system gave a coefficient of variation of about f 2 for 18 individual reactions (also for only six), which is much superior t o the SI method and nearly approaches the in-
ANALYTICAL CHEMISTRY, VOL. 51, NO. 9, AUGUST 1979
50
-
40 -
-2
.->
-
30-
--C
c
.-01 2 0 A
~~
30 40 50 60 Diethyiirolurninol (pM) Flgure 4. Comparison of the RMF system with the SI system for monitoring chemiluminescence reactions. Diethylisoluminol at the indicated final concentrations in the cell was oxidized with the RMF system and the light intensities were recorded as described under "Experimental" (A). Each data point is the result of one measurement. Reaction mixtures (150 pL) containing indicated levels of diethylisoluminol received a single automatic injection of H,Op to produce light as described in the Experimental section. The peak light intensities from three individual reactions were averaged for each data point (0) 10
20
Table I. Reproducibility of Chemiluminescence Measurementsa light standard 0.9
single injection system automatic rapid mixing hand flow system injection injection 2.0
38
8
a Each value represents the coefficient of variation (%) of the mean light intensity obtained with 18 identical reactions. The concentrations of chemiluminescent com-
pound were chosen t o produce similar light intensities with each method of measurement (also the light standard) to allow direct comparison of reproducibility. strument variability of hO.970 (Table I). T h e light standard described in the Experimental was measured repeatedly and the coefficient of variation is presented for reference. Chemiluminescent reactions were carried out with diethylisoluminol (30.8 pM final concentration) in the RMF system as described in the Experimental and 18 ten-second measurements were obtained. The mean light intensity was 22.6 and a background signal (absence of chemiluminescent compound) was generally 0.5. Similar chemiluminescence measurements were performed with the S I system utilizing automatic addition of HzOzas described previously ( 4 ) or by hand from a 25-pL Hamilton syringe as described in the Experimental.
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The enhanced reproducibility may partially be based on reaction mechanism. Since microperoxidase is destroyed by H,02, true catalysis by the haem moiety is observed only in the initial part of the reaction (10). Therefore, at steady-state chemiluminescence, sufficient rapid displacement of cell content may eliminate destruction of microperoxidase within the flow cell and thereby reduce variability in measurements. The residence time of reagents in the flow cell was about 6 ms. Other flow cells typically have usually greater than 50 ms (5). Such residence times are too long to take advantage of potential sensitivity and reproducibility of flow system with fast reactions. Precision in chemiluminescence measurements is related to speed of light production and to the reagent mixing system. The SI system gives poor reproducibility when light is produced rapidly because bubbles form in the solution and scatter light. Measurement of total light does not improve reproducibility in this case ( 4 ) . An instrument with high speed injection below the reaction surface may provide rapid mixing and improved reproducibility. We have taken an alternative approach and shown that variability in highly sensitive chemiluminescence measurements can be greatly reduced with an instrument based on rapid mixing and short cell residence time; e.g., at maximum light intensity, values became independent of flow rate. Only small quantities of commercially available, inexpensive reagents are required since the flow time for steady-state chemiluminescence is short (less than 10 5). Furthermore, detection reactions at pH 8.6 to 13 possess similar sensitivity ( 4 ) and the RMF system was also successful in brief tests in 7 5 mM barbital, pH 8.6. Although the system described here was designed for the specialized purpose of monitoring competitive protein binding reactions, other types of chemiluminescence measurements may benefit from a similar approach.
ACKNOWLEDGMENT We thank Frances M. Yeager for performing some of the chemiluminescence measurements.
LITERATURE CITED Schroeder, H. R.; Vogelhvt, P. 0.; Carrico, R. J.; Boguslaski, R. C.; Buckler, R. T. Anal. Chem. 1976, 4 8 , 1933-1937. Schroeder, H. R.; Bogushski, R. C.; Carrico, R. J.; Buckler, R. T. Methais Enzymol. 1978, 57, 424-445. Schroeder, H.R.; Yeager, F. M.; Boguslaski, R. C.; Vogelhut, P. 0. J . Immunol. Methods 1979, 25, 279. Schroeder, H. R.; Yeager, F. M. Anal. Chem. 1978, 50, 1114-1120. Seitz, W. R.; Hercules, D. M. Anal. Chem. 1972, 4 4 , 2143-2149. Seitz, W. R.; Neary, M. P. In "Methods of Biochemical Analysis", Vol. 23, Glick. D., Ed.; John Wiiey and Sons: New York, 1976; Vol. 23, pp 161-186.
Stieg, S.; Nieman, T. A. Anal. Chem. 1978, 50, 401-404. Sheehan, T. L.; Hercules, D. M. Anal. Chem. 1977, 49, 446-450. Bostick, D.T.; Hercules, D.M. Anal. Chem. 1975, 4 7 , 447-452. Kremer, M. L. Natufe (London) 1965, 205, 384-385.
RECEIVED for review March 7,1979. Accepted April 11, 1979.
Pulse Dampener for High Pressure Liquid Chromatography John G. Nikelly" and Dominic A. Ventura Department of Chemistry, Philadelphia College of Pharmacy and Science, Philadelphia, Pennsylvania
In the past two years mechanical reciprocating piston pumps have become the most widely used type of pump in high pressure liquid chromatography, owing to several advantages such as having a small internal volume and placing no restriction on the size of the solvent reservoir.
19 104
However, it should be noted that virtually all of the new pumps that are specifically designed for HPLC are of the multiple-head or multiple-action type. In addition, many of the multiple pumps are equipped with expensive features designed to further reduce or totally eliminate the pulsations
0003-2700/79/035 1-1585$01. O W 0 @ 1979 American Chemical Society
