1721
Anal. Chem. 1981, 53, 1721-1723
interval from 3 M HF/0.625 M HNOBto 21.6 M HF/1.5 M HNO,; and in Figure 4 from the mixture of hydrofloric acid and nitric acid 1:l between 2 M HF/1.25 M HNOB and 12 M HF/7.5 M "0,. The quoted values are the average of a minimum of two determinations. For an adsorption of 50% (log D F= 1.7) the relative standard deviation is *3%. The results in Figure 1 are in accordance with the data published by Faris for dynamic conditions (1). Only for palladium, platinum, gold, and mercury differing D values have been obtained, presumably due to dzferent experimental conditions. Rather unreproducible results were obtained for the adsorption of silver ,and antimony. These results are not given in detail. For niobium, the observed increase in D with increasing acid concentration can be attributed to the disappearance of oxyfluoro complexes. The comparison of results given in Figures 1-4 indicates that with increasing fraction of nitric acid there is a continous decrease of the D value of all elements tested. This can be explained by the formation of less charged and less adsorbable anionic species in the presence of nitric acid. These distribution data enable one to work out useful separation procedures. For instance, on their basis a rapid
separation procedure has been developed for the radiochemical neutron activation analysis of high-purity niobium involving short-lived indicator radionuclides (3).
LITERATURE CITED (1) Faris, J. P. Anal. Chem. 1960, 32, 520-522. (2) Faris, J. P. Anal. Chem. 1064, 36, 1157-1158. (3) Faix, W. G.; Caietka, R.; Krivan, V. Anal. Chem. 1981, 53, 1594- 1598.
Werner G. Faix Rostislav Calet ka Viliam Krivan* Sektion Analytik und Hochstreinigung Universitat Ulm Oberer Eselsberg N26, D-7900 Ulm Federal Republic of Germany RECEIVED for review March 24,1981. Accepted May 26,1981. This project was financially supported by Bundesministerium fur Forschung und Technologie, Bonn. Irradiation facilities for the production of radionuclides were provided by Kernforschungszentrum Karlsruhe.
Comments on Noise and Digital Resolution in a Microprocessor-Controlled Spectrophotometer Sir; We would like to a.dd to the paper by Kaye and Barber ( I ) . They made the remark that surprisingly they found the temperature coefficient of the photomultiplierswas somewhat less than the figure given by the manufacturer, Hamamatsu Corp. This is not especially surprising to us. The main reason being that all of our data are taken from large samplings and in general indicate a worse case situation. That is true not only for temperature coefficients but also for spectral response curves. In actuality, there is a resonable variance tube to tube, since the cathodes of photomultiplier tubes tend to be both interference filters and plhoto emissive devices. Each layer of the film tends to be quite thin and not precisely controlled in thickness. In recent years we have found a tendency for the detectors to become more and more uniform. This is probably the result of better control over the materials used in making the tube, plus a better environment within our factory situation. But, the user and especially the design engineer should take note
of the fact that the data shown in detector manufacturer's catalogs is statistical in nature and does not represent any particular tube. For this reason, for many years Hamamatsu has taken a very strong negative stand utilizing photomultiplier tubes as standards over a long period of time. Applications such as the one described by Kaye, which rely on relatively short term stability are within the state of the art.
LITERATURE CITED (I) Kaye, Wibur; Barber, Duane Anal. Chem. 1981, 53, 366-369.
R. Eno Hamamatsu Corporation 420 South A~~~~~ Middlesex, N~~ jersey 08846 RECEIVED for review March 4,1981. Accepted April 10,1981.
AIDS FOR ANALYTICAL CHEMISTS Colorimetric Measurements in a Liquid Scintillation Counter Martin W. Heitzmann" sind Leonard A. Ford Division of Drug Chemistty, Food and Drug Administration, Washington, D.C. 20204
The concept of making high-precision photometric measurements by using a radiosotopic light source was first reported by Ross ( I ) . The instrument used in his experiments was a hybrid constructed from portions of a liquid scintillation counter (LSC), a multichannel pulse height analyzer, and a
custom-made sample chamber containing standard 1-cm cuvettes for the light source and sample holder. In an earlier paper (21, Ross described the use of an isolated internal standard cell to distinguish between the contributions of chemical and color quenching to total quenching. By se-
This article not subject to U S . Copyright. Published 1981 by the American Chemical Society
1722
ANALYTICAL CHEMISTRY, VOL.
53, NO.
11, SEPTEMBER 1981
Table I. Spectral Parameters in Colorimetric Measurements compd K,Cr,O, Co(NO,),.GH,O
Cr(OAc),.H,O
C?
mol/L
Amax,
nm
4.43 X 370 10-5 0.1030 510 0.0253 430
b
AC
ed
kIe
0.633
1.43 X 104 6.69 21.6
1.55 X 104 5.12 272.4
0.690 0.546
#-
+
ci
i , 3
Molar concentration used in absorbance reading. Absorption maximum. Absorbance at A, for 1 cm path length. Molar absorptivity. e Displacement sensitivity constant.
- 45 mm
length flame sealed ampoule
lective integration of these two concepts, we have been able to parallel Ross’ photometric measurements using an unaltered commercial LSC with a combination light source/sample cell made from a standard 20-mL glass liquid scintillation vial. EXPERIMENTAL SECTION Apparatus. The liquid scintillation counter used was a Beckman LS-133, equipped with two variable “isosets” for upper and lower discriminator settings. These “isosets” were 10-turn potentiometers which had a reproducibility of 0.1%. The absorption spectra were obtained from a Bausch and Lomb 505 recording dual-cellspectrophotometerusing 1-cmVycor cells. All pH measurements were made with a Beckman Zeromatic I1 pH meter. Reagents. An aqueous solution of ammonium pertechnetate, labeled 0.503 mCi/mL, was obtained from Chemical and Radioisotope Division, International Chemical and Nuclear Corp., Irvine, CA, and was used as received. Methyltricaprylammonium chloride (MTC) (Aliquat-336) was obtained from A m o u r and Co., Kankakee, IL, and was used as received. The concentrated scintillant solution was Spectraflor, obtained from Amershaml Searle Corp., Des Plaines, IL (Amersham,Arlington Heights, IL). Reagent grade potassium dichromate, cobalt nitrate, and chromium acetate were used without purification to prepare stock solutions. The concentrations of the cobalt nitrate and chromium acetate aqueous working solutions and the potassium dichromate pH 8 buffered working solution are given in Table I, along with the chemical formulas for these compounds. All potassium dichromate stock solutions were made to pH 8.0 with sodium bicarbonate and, because an error caused by pH change could be introduced by diluting the potassium dichromate solution with water, all dilutions of the dichromate stock solution were made with pH 8 bicarbonate solution. Preparation of Radioactive Light Source. The radioactive ammonium pertechnetate, light source was prepared from (99’~) MTC, and Spectraflor. The pertechnetate was extracted from the aqueous solution with a solution of MTC in toluene (1g/100 mL) (3). The desired activity per microliter was obtained by evaporating the toluene. A concentrated scintillant solution was added to the evaporated pertechnetate extract to obtain the final solution. An aliquot of this solution containing approximately 3-5 FCi of technetium was transferred to a 1 / 8 in. (0.32 cm) 0.d. thin-walled Pyrex tube. The solution was frozen with dry ice, and the tube was flame-sealed under vacuum (Figure 1). Procedure for Colorimetric Measurement. All of the colorimetric measurements were made and the concentrations of the solutions (Table I) were fitted to standard curves by the following procedure. Vials were always filled so that the liquid was above the lower edge of the Teflon collar with the cap assembly screwed in place (Figure 1). The channel width was kept constant throughout the experiment at about 6% of maximum width (upper and lower discriminator settings of 3.6 and 3.0, respectively). Time was preset to give 0.2% error in counting statistics for the blanks. Blanks were counted before and after changing from one colored species to another. All samples were counted in sequence from lowest to highest concentration. Concentrations of solutions used for spectrophotometric measurements were adjusted to give absorbance values between 0.4 and 0.8. Absorbances were measured on a double beam re-
3.2mm
Flgure 1. Combination llght source/sample cell made from a standard 20-mL glass liquid scintillation vial. I06
105
.-c C r
104
IL
\ v)
c
5 io3 8 e 102
IO Pulse Height p pulse-height distrlbutions.
Flgure 2. Graphic representation of
cording UV/visible spectrophotometer in 1-cm cells with water as a blank. RESULTS AND DISCUSSION The theoretical principles involved are fully developed by Ross (1) and discussed by Schram (4). However, a brief summary of those principles will make it easier to follow the description of our work. The phenomenon of color quenching can be used t o determine the concentrations of colored solutions. It has been established that the p energy distribution curve, as depicted by photon intensity in the liquid scintillation process, approximates an exponential function over a major portion of the p energy spectrum for certain radionuclides. In this well-behaved region, the distribution is a linear function of the absorbance of any colored filter interposed between the source and the detector. By the interposition of filters of increasing opacity, a family of lines that seem to rotate about a common focal point is obtained. If a semilog plot of an unfiltered distribution is made by using that focal point as zero on the pulse height axis, and a total count of 10 is arbitrarily selected as the intercept Po,the dotted line shown
1723
Anal. Chem. 1981, 53, 1723-1725
in Figure 2 is obtained. When a colored filter, C, is introduced between the scintillating light source and the detector, the solid line interacting a t PC is obtained. If discriminators d l and d2 are added to the system, the shaded areas rc and PO will represent total counts detected from the light source, with and without filter, respectively. A more detailed discussion is given by Ross (1). The concentration/diisplacementrelationship developed by
Ross
R = log (ro/rc) = kc
(1)
where R is the displacementfactor, ro is the count in the blank, rc is the count in the sample, k is the absorption constant for the system, and c is the molarity of the absorbing species, is used here with one significant change. The sensitivity factor, k', used in this study is empirical rather than derived from the theoretical principle used by Ross. This difference in the respective sensitivity factors is due to a combination of elements. In our instrumentation (a) the sample symmetrically surrounds the light source; (b) the light is detected by twin photomultipliers; and (c) the resulting signal is processed through coincidence-circuitry. It was not our intention to repeat the Ross experiment in its entirety; it was only necessary to demonstrate that we could generate adequate standiwd curves and compute k'values for a range of colored species. We found that a1 linear relationship between concentration ( c ) and displacement (R) exists for these solutions which ranged in color from orange to green. The k ' values for these salts are listed in Table I, which contains both experimental and calculated values. The expected decrease in sensitivity in going from the red to the blue end of the absorption spectrum is due to both the fluorescence spectrum of the scintillant and the response curve of the phototubes. Although there is no theoretical basis for comparing molar absorptivity (e) values with sensitivity factor (k? values, it is noteworthy that they are of the same order of magnitude, except in the case of chromium acetate. The apparent anomalous behavior of the latter may be due to its double absorption maxima shown in Figure 3. The advantages of thiri type of photometric measurement over conventional photometry were discussed in detail by Ross and include: (a) accuracy and precision limited only by statistical considerations of iradioactivity counting; (b) ability to handle very dilute to very concentrated samples with equal reliability; (c) stability of the light source; and (d) ability to feed digital output directly into a computer without analogue to digital conversion.
IW
sa0
\
/
I
0.2
110
1w
410
490
1110
e10
e10
Mo
e90
Wavebqih ( nm )
Flgure 3. Absorption spectra of K2Cr207,C~(OAC)~, and Co(NO&.
The inherent limitations of our sample cell are comparable to those of the Ross system and are subject to the same corrective measures, e.g., the introduction of narrow band-pass filters around the light source to minimize interferences from other colored species. The loss in sensitivity resulting from the decreased light output is readily compensated for in either system by increasing the concentration of the radioisotope in the light source. Our light source/sample cell, however, has its own unique advantages: (a) it requires no modification of the LSC; (b) it is inexpensive and simple to fabricate; (c) its practical visible range can be altered simply by changing to a different scintillant system; and (d) it should have applications in numerous instances, such as in differential reaction rate studies, where it is very difficult to use conventional photometry. The most attractive feature of this light source/sample cell is that it permits high-precision measurements to be made a t low cost with an unaltered LSC. ACKNOWLEDGMENT The authors thank Armour and Co., Kankakee, IL, for their generous gift of Aliquat-336.
LITERATURE CITED (1) Ross, H. H. Anel. Chem. 1966, 38, 414-420. (2) Ross, H. H. Anal. Chem. 1965, 37, 621-623. (3) . . Salarla. G. 6.: Rulfs. C. L.: Elvina. P. L. Anal. Chem. 1983, 35. 983-985. (4) Schram, Eric "Organic Sclntlllatlon Detectors"; American Elsevier: New York, 1963; pp 21-28.
RECEIVED for review March 11,1981. Accepted May 18,1981.
Electronic Ruler for Digitizing Data S. R. Inman," S. A. Sibbald, and R. B. McComb Clinical Chemistry Laboratoty, Department of Pathology, Hartford Hospital, Hartford, Connecticut 06 1 15
A major advantage of high-performance liquid chromatography (HPLC) is that niultiple components of interest are analyzed within any one run. Under ideal conditions, each component is identified by the retention time of its associated chromatographic peak andl is quantitated by the peak height or area of this same peak. However, as the number of components increase, the qutintification becomes tedious and significantly adds to the total analysis time. The use of on-line integrators coupled with automated data processing apparatus
is one solution to this problem. An alternate solution, and one which is being used successfully in our laboratory, involves measuring the peak heights of the pertinent chromatographic peaks by means of a caliper coupled to a slide wire potentiometer and feeding the digitized signal to a small programmable calculator. All data reduction is performed by the calculator, and the results are printed on a tape. Our HPLC system is being used for the analysis of five antiepileptic drugs (AED).
0003-2700/81/0353-1723$01.25/00 1981 Amerlcan Chemlcal Soclety
