Table 111. Relative sensitivities are compared for lead, bismuth, selenium, thallium, and tellurium when alone and in the presence of nickel, a nickel-base alloy, an iron-base alloy, or a cobalt-base alloy. The magnitudes of the interferences were greatest for selenium, where a cobalt alloy matrix enhanced the selenium signal by a factor of two. It is interesting to note that there was not a large difference in sensitivity for any of the elements in nickel or in a nickel alloy. For most precise work, standards must be similar in composition to the samples. Purge Gas Flow Rate. The flow rate of argon through the graphite tube was not critical. In the cases of lead, thallium, and tellurium, significant improvements in detection limits were obtained when the purge gas flow was interrupted for a short time during the char cycle. Spectral Band-Pass. The effects of spectral band-pass
on signal-to-background ratios were measured for each of the five analyte elements. The effects were negligible except in the case of bismuth, where a smaller band-pass partially resolved the 223.1- and 222.8-nm lines and improved the signal-to-background ratio by less than a factor of two. Comparative Results. The four nickel-base reference alloys described previously were analyzed by the graphite furnace-atomic absorption procedure. Table IV lists a comparison of the values obtained by this technique with values obtained by other methods. The precision of the method was evaluated by analyzing ten separately weighed portions of a nickel-base alloy. The results, based on single absorbance measurements, are tabulated in Table V. Received for review January 14, 1974. Accepted April 19, 1974.
Simultaneous Determination of Electrolytes in Serum Using a Vidicon Flame Spectrometer K. W. Busch, N. G. Howell, and G. H. Morrison' Department of Chemistry, Cornell University, Ithaca, N. Y. 14850
The rapid simultaneous determination of sodium, potassium, and calcium in blood serum is described using a recently developed vidicon flame spectrometer. The system, which includes a silicon-intensified target vidicon tube, monitors a 40-nm spectral window. The simultaneous analysis is accomplished by direct injection of 200 pI of serum into a nitrous oxide-acetylene flame, and multichannel monitoring of the spectral region 807 nm to 847 nm. Results for control serum and clinical samples indicate an accuracy and precision of better than 2 % .
Requests for r e p r i n t s s h o u l d be addressed t o G. H.M o r r i s o n , D e p a r t m e n t of C h e m i s t r y , C o r n e l l U n i v e r s i t y , Ithaca, N.Y. 14850.
Speed, accuracy, and precision are the three most critical factors in clinical analyses. The clinical methods currently in use for the determination of sodium, potassium, and calcium suffer disadvantages in that they require some form of sample pretreatment or dilution. Sample pretreatment can adversely affect the accuracy of a sodium, potassium, or calcium determination through contamination of the sample from reagents or glassware as well as degrade the precision through unavoidable manipulative errors. T o provide the required speed using these methods, various means of automation have been utilized, some of which are quite complicated and are prone to various mechanical difficulties. The use of flame techniques for the determination of individual electrolytes in body fluids has been described by many investigators using conventional detectors. We have recently described ( 4 ) a vidicon flame spectrometer which permits simultaneous multielement analyses by flame emission. The vidicon detector simultaneously monitors a given range of wavelengths, the information being acquired in 500 electronic channels. In comparison with previous multielement systems ( 5 ) ,this system is inherently versatile and simple and shows considerable potential for clinical analyses. This paper describes the application of the vidicon flame spectrometer to the simultaneous determination of sodium, potassium, and calcium in serum. Serum samples are introduced into a flame source by injection of microvolumes of sample without dilution or pretreatment using a hypodermic syringe. The transient signals produced by flame emission are dispersed using a grating monochromator and detected using a silicon intensified target vidicon tube.
Ronald L. Searcy, "Diagnostic Biochemistry," McGraw-Hill Book Co., New York, N.Y., 1969. John Lott, CRCCrit. Rev. Anal. Chem., 3 ( l ) ,4 1 (1972). C. R. Kleeman, S. B. Massry, and J. W. Coburn, Calif. Med., 114, 16 (1971).
(4) K. W. Busch, N. G. Howell, and G. H. Morrison, Anal. Chem., 46, 575 (1974). (5) K. W. Busch and G. H. Morrison, Anal. Chem.. 45, 712A (1973).
The determination of sodium, potassium, and calcium in human blood serum and urine is of considerable importance to the physician in assessing the electrolyte balance of the patient. Hyponatremia and hypocalcemia are frequent findings, especially in those who are acutely ill ( I ) . For example, chronic illness from rickettsial disease, meningitis, and pulmonary tuberculosis all exhibit hyponatremia (I).Patients exhibiting toxemia of pregnancy usually exhibit serum sodium levels 4 to 8 mequiv/l. lower than normal pregnant females (I).Patients suffering from Addison's disease are prone to hyponatremia and hyperkalemia because the kidneys fail to conserve sodium and water but retain potassium ( I ) . Hypercalcemia has been observed in multiple myeloma, metastatic carcinoma of bone, and bronchocarcinoma (2). Hypocalcemia has been observed in hypoparathyroidism (3).Acute hypocalcemia results in tetany leading to convulsions, laryngeal spasms, and death.
ANALYTICAL CHEMISTRY, VOL. 46, NO. 9 , AUGUST 1974
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RESERVOIR
I -
Table 11. Analytical Data on the CDC Reference Sera
SAMPLE INLET
+Fj i
Standard
0 )
BURNER
MONOCHROMATOR
LENS
SLIT
Sodium," mequiv,l.
1
124
3 5
138
Potassium: mequiv, 1.
154
a CDC Reference laboratory values. trometry values.
Calcium," mg dl
2.74 4.66 6.71
5.16 8.97 13 .O
NBS isotope dilution mass spec-
Figure 1. Vidicon flame spectrometer system
Table I. Experimental Facilities Burner
E x t e r n a l optics
E n t r a n c e slit Monochromator
Detector
Optical multichannel analyzer Readout Flow m e t e r s
Varian T e c h t r o n 5-cm high-solids slot b u r n e r for n i t r o u s oxideacetylene. Five-cm d i a m e t e r Supracil lens w i t h 12.5-cm focal l e n g t h . L e n s s t o p p e d d o w n t o 2.6-cm diameter. Jarrell-Ash M o d e l 12-080 variable 0-2000 pm s t r a i g h t edged slit. Jarrell-Ash, M o d e l 82-000, 0.5-m E b e r t m o u n t i n g scanning monoc h r o m a t o r w i t h 590 grooves ' m m g r a t i n g blazed f o r 4000 A. Reciprocal linear dispersion 32 8, m m in the first order. Silicon Intensified T a r g e t Vidicon, M o d e l 1205D, SSR Instrum e n t s Co., S a n t a M o n i c a , Calif. M o d e l 1205A, SSR I n s t r u m e n t s Co., S a n t a Monica, Calif. T e c h t r o n i x Oscilloscope, M o d e l 604 M o n i t o r . B r o o k s Full-view R o t a m e t e r s calibrated f o r n i t r o u s oxide a n d acetylene, B r o o k s I n s t r u m e n t
co. Syringe
H a m i l t o n Co. microliter syringe Nn. 725.
EXPERIMENTAL Apparatus. The experimental facilities are described in Table I. Vidicon Flame S p e c t r o m e t e r . Figure 1 shows a schematic diagram of the vidicon flame spectrometer, which consists of a nitrous oxide-acetylene flame source, external optics, monochromator, silicon intensified target vidicon tube (SIT), optical multichannel analyzer (OMA), and oscilloscope display. The S I T vidicon was mounted on a 0.5-m Ebert monochromator equipped with a 590 groove-per-millimeter grating to provide a wavelength window of 40 nm across the tube. A mounting plate similar to the one described previously ( 4 ) allowed the dispersed spectral lines to be focused on the SIT fiber optic faceplate. A bayonet mounting variable straight-edged slit was used for the entrance slit. The optical multichannel analyzer has two separate memories, which allow the storage of both a data spectrum and a blank spectrum; each memory contains 500 words. The contents of a given channel are displayed digitally on the display panel of the console, and on the cathode ray tube as an intensified cursor spot. An arithmetic unit permits the channel-by-channel subtraction of memory B from memory A. Measurement of the integrated intensity of time-varying signals may be accomplished by accumulating a preset number of frame scans in memory. The detailed operation of the optical multichannel analyzer has been previously discussed (1). S a m p l e Introduction. The sample introduction system, shown in Figure 1, is used to introduce small volumes of serum directly in a high-solids nitrous oxide-acetylene slot burner, without sample pretreatment or dilution. The injection port is fabricated from Plexiglas and utilizes a silicone rubber septum which permits injection of the sample with a microsyringe in a manner similar to 1232
that used in chromatography. The liquid reservoir is connected to the injection uort with a length of 0.062-inch i.d. uolvethvlene tubing (Clay Adams Intramedic, Cat. No. P E 205). length of 0.034inch i.d. polyethylene tubing (Clay Adams Intramedic, Cat. No. PE90) is used t o connect the injection port to the burner. The reservoir is fabricated from a 1-liter polyethylene wash bottle. A small hole made in the cap allowed the polyethylene tubing to be cemented in place with silicone rubber cement. When in use, the bottle is hung about 60 cm above the burner in an inverted position from a stand, in a manner similar to that used in giving intravenous transfusions. In this position, air is automatically admitted into the bottle through the wash bottle nozzle as the liquid flows out the polyethylene tubing to the injection port. The bottle is filled with an 1160 Fg ml-' solution of cesium chloride as an ionization buffer. A similar sample introduction system has been described by Winefordner and coworkers (6). Filters. A filter is required t o attenuate the intensity of both the Na 818.3-819.5 nm lines and the Ca 422.7 nm line without reducing the intensity of the K 404.4-404.7 nm lines. A filter which accomplishes this was fabricated using two silica 1.0-cm path length spectrophotometer cells mounted in tandem directly in front of the entrance slit. One cell was filled with an aqueous copper sulfate solution and the other filled with a solution of iodine in carbon tetrachloride. The copper sulfate solution was prepared by dissolving 8.31 grams of C u S 0 ~ 5 H 2 0in water and diluting this solution to 500 milliliters. This solution had an absorbance of 0.88 with respect to water a t the sodium 819.5 nm line when using 1.0-cm path length cells. The iodine-carbon tetrachloride filter was prepared by making a saturated iodine solution in carban tetrachloride and diluting this solution with carbon tetrachloride until the resulting solution had an absorbance of 1.33 with respect to carbon tetrachloride at the calcium 422.7 nm line when using 1.0-cm path length cells. Standards. Analyzed bovine serum samples at three concentration levels (for sodium, potassium, and calcium) were supplied by the Center for Disease Control (CDC), Atlanta, Ga., and used as standards. Streptomycin, penicillin, and fungizone were added to the serum samples a t the CDC to prevent any bacteriological action that might lead to analytical errors. The concentrations of each element in each bovine serum standard are given in Table 11. Procedure. The anaIytical curves for sodium, potassium, and calcium were prepared simultaneously by injecting 200 1 1 of each bovine serum standard into the flame with a microsyringe (Hamilton Co., No. 725), accumulating the signal for 400 accumulation cycles (12.8 sec), and storing the result in memory A. The blank solution of CsCl (1160 ppm), which was continuously being aspirated into the flame from the reservoir, was accumulated for an equivalent amount of time, and the result stored in memory B. The A minus B mode was selected to display the channel-by-channel difference between the two memories. Peak heights of spectral lines were recorded digitally from the digital display by moving the cursor to the given peak. Adjacent background to a spectral line was measured by selecting the summation mode, and recording the average of ten adjacent channels on the digital panel display. The average background-corrected peak height for two injections of each standard was plotted us. concentration to give the analytical curves. Serum samples were analyzed by injecting 200-p1 volumes into the flame without any prior pretreatment or dilution in a manner analogous to that used for the standards. Each sample was run twice and the average background-corrected peak height was compared with the calibration curves to give the concentrations of sodium, potassium, and calcium.
k
(6) J. R. Sarbeck, P. A. St. John, and J. D. Winefordner, Microchim. Acfa 1 Wien], 1972, 55.
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 9, AUGUST 1974
Si wafer
RESULTS A N D DISCUSSION
,D-lvDe
\
semiconductor
SIT Vidicon. The ability to perform simultaneous multielement analysis with this system depends on the use of the vidicon detector and the associated optical multichannel analyzer. The standard UV sensitive image vidicon used in our initial description of the vidicon flame spectrometer ( 4 ) was unsuitable for the present study because it was unable to detect the relatively weak potassium lines in the second-order spectrum employed in the chosen window. The SIT tube is essentially similar to the UV sensitive image vidicon with the addition of a photocathode intensifier stage prior to the silicon target as shown in Figure 2. Dispersed spectral lines focused on the fiber optic faceplate cause photoelectrons to be emitted from the S-20 photocathode surface. These photoelectrons are collimated by the curved photocathode surface and internal focusing electrodes to produce an electron image on the silicon target which corresponds as closely as possible t o the optical image focused on the faceplate of the tube. Because of the accelerating potential, the emitted photoelectrons stiike the target with energies on the order of 9 KeV, where they produce holes and electrons in the n-type silicon wafer. The SIT tube is more sensitive by approximately two orders of magnitude than the UV sensitive image vidicon because hole production resulting from electron impact is increased over that produced by direct photon absorption. By use of the S I T tube, the weak second-order potassium lines could easily be detected a t the concentrations employed in this study. Selection of Analytical Conditions. In single element determinations, the analytical conditions are optimized to determine the given element with high accuracy and precision. The compromise analytical conditions required in multielement analysis must be carefully selected t o determine the given elements simultaneously with the highest accuracy and precision obtainable. Table I11 summarizes the analytical conditions used for this method. Windou: Selection,. Simultaneous analysis of sodium, potassium, and calcium in serum using the vidicon flame spectrometer is based on a “window” concept-Le., the range of wavelengths simultaneously monitored by the tube. An optimum window for these elements in serum and urine was achieved from approximately 807 nm to 847 nm. This window allowed the simultaneous determination of sodium, potassium, and calcium a t the levels encountered in human serum or urine without any sample pretreatment or dilution. Since sodium is present in serum a t concentrations on the order of 3000 pg ml-l, the 818.3-819.5 nm sodium douhlet provided adequate intensity for direct analysis. Since these lines are not resonance lines, the severe self-absorption which would be associated with the conventionally used resonance lines was avoided. The use of the calcium 422.7 nm resonance line and the potassium 404.4-404.7 nm resonance doublet, which are observed in the second order ( K 808.8-809.4 nm, Ca 845.4 nm) allowed the simultaneous determination of sodium, potassium, and calcium in this 40-nm window. It should be noted that when using a sensitive detector such as the SIT tube, as narrow a wavelength range as possible should be employed to minimize the possibility of detector saturation by intense flame bands and other intense lines present in the analyte spectrum. For example, if the potassium and calcium lines were observed in the first order, a window extending from 404.4 nm to 819.5 nm would have been required. In addition to the poor resolution obtainable with such a wide window, large portions of the window would saturate the detector because of the
GU1
Figure 2.
beam
Silicon-intensifiedtarget vidicon detector
Table 111. Analytical Conditions
Flame
Ionization buffer Sample size Slit width Spectral window Accumulation cycles
Fuel: CzH?, 8 lbs in.-z, 3500 cm3 min-1 Oxidant: N,O, 30 lbs in.-*, 4750 cm3 min-1 Region viewed: 22 mm above burner top 1160 ppm Cesium 200 pl 60 pm
807-847 nm 400 Frame scans
presence of intense lines such as the potassium 766.5-769.9 nm doublet, the sodium 589.0-589.5 nm doublet, and the CaOH band a t 622 nm. The presence of these intense radiations within the window would saturate the remaining regions within the window by the “blooming” or spreading of the saturation to adjacent channels, making the entire window useless. Since the weakest lines in the selected window were the potassium 404.4-404.7 nm doublet in the second order, the analytical conditions were adjusted to maximize the intensity of these lines. The intensity of the potassium lines was increased to adequate levels by opening the slit to 60 pm. This raised the intensity of the sodium and calcium lines to a level where the detector was saturated. The absorption filter described in the experimental section was employed to reduce the intensity of the sodium and calcium lines without concomitantly reducing the intensity of the potassium lines. The aqueous copper sulfate solution was used to attenuate the sodium lines, while the iodine-carbon tetrachloride filter was used to attenuate the calcium line. Adjustment of the concentrations of these solutions al!ows for independent attenuation of the sodium and calcium signals. A typical spectrum for a 200-4 injection of serum is shown in Figure 3. The necessity for the use of filters is due to the somewhat limited dynamic range of the vidicon tube in the realtime mode. The vidicon tube operates on a charge ;t orage principle ( 4 ) in which an electron beam, which scans the target in a raster pattern, deposits a given charge on the tube target. If a spectral line of sufficient intensity is imaged on the tube target, the charge deposited by the electron beam may be completely depleted before the electron beam returns to the given target element. Under these circumstances, the tube is “saturated” a t this location. The dynamic range of the system may be extended by means of computer control of the scanning electron beam. By this means, regions of the target subject to high intensities are scanned by the electron beam more frequently-ie., before all the deposited charge is depleted-than those regions exposed to lower intensities. This differential scanning technique should allow the analyst to increase the apparent dy-
A N A L Y T I C A L CHEMISTRY, VOL. 46,
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NO. 9. AUGUST 1974
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SERUM
v u C t
0 0
--
Figure 4. Transient injection pulse
ii
Table IV. Analysis of Control Serum
v u
rwn lw n
5E Plc 00
Pe YY
Certificate
I*
40.0nm
I.
Figure 3. Spectrum of serum for 200-plinjection
namic range of the tube without resorting to optical filters. The optical multichannel analyzer used in this study, however, did not include computer control of the scanning electron beam, so filters were necessary. Flame. The nitrous oxide-acetylene flame was selected to avoid the well known phosphate interference on the emitted calcium intensity. The optical axis was adjusted so that the flame was observed a t 22 mm above the burner to avoid the lateral diffusion interference of phosphate on calcium ( 7 ) .Under these conditions, aqueous solutions of calcium containing one hundred times the normal phosphate level in serum gave the same signals as similar solutions without phosphate. In addition, the high temperature nitrous oxide-acetylene flame used in this method produces a sufficiently strong signal for the potassium lines a t the concentration levels of serum. Air-acetylene or air-propane flames normally used in flame clinical methods did not produce measurable signals for these potassium lines using the vidicon detector. Accumulation Time.The signal produced when a sample is injected into the sample introduction system is a timevarying transient pulse. Figure 4 shows the variation of analyte intensity as a function of time when a 200-p1 sample of serum was injected into the flame. This trace was obtained by setting the cursor on the calcium 422.7-nm line and monitoring the growth and decay of the calcium intensity with an oscilloscope. The quantity of analytical interest is the total analyte intensity for a given injection-Le., the time-integrated intensity over the duration of the pulse. If a real-time detector such as a multiplier phototube is used with this system, the maximum of the transient pulse must be taken as the analytical signal unless some additional device for signal integration is used. If such a device is unavailable, the analytical results depend on the reproducibility of the transient pulse, which depends on many factors. The vidicon tube, in contrast, is an integrating detector ( 4 ) , which continually integrates the intensity of light striking the tube. The integral of intensity for a given target element is read once every 33 msec when the electron beam addresses that target element and erases the accumulated positive charge. To integrate a time-varying intensity for a period longer than 33 msec, the accumulation mode on the OMA is selected and the signal is accumulated in memory A for a preset number of frame scans. ( 7 ) A. C. West, V. A. Fassel, and R . N. Kniseley, Anal. Chern., 45, 2420 (1973).
1234
Sodium, mequivjl. RSD, % Dev, % Potassium, mequivll. RSD, % Dev, % Calcium, me/dl RSD' % Dev, %
CDC standards
Aqueous standards
141 i 0 . 9 0.64 ...
139 i 0 . 5 0.36 1.42
149 10 . 7 0.47 5.67
4 . 3 + 0.06 1.39
4 . 2 i0.05 1.19 2.23
4 . 5 i0.02 0.44 4.65
9 . 5 +0.16 1.68
9.6 1 0 . 1 7 1.77 1.05
11.3 1 0 . 2 8 2.48 18.95
...
...
This causes the time integrated signals for each target element for each frame scan of the electron beam to be deposited in their respective locations in memory. Thus the final peak intensity of the spectral lines displayed on the oscilloscope screen after a preset number of frame scans or accumulation cycles is the sum of the individual intensity integrals for each frame scan or the total integrated intensity of the transient pulse. An accumulation time of 12.8 sec or 400 frame scans was chosen to ensure that the vidicon was integrating the intensity for the entire duration of the sample emission pulse produced by injecting 200 p1 of serum. Analytical Results. Table IV shows the results of the simultaneous analysis of a control serum (Lab-Trol, Dade Reagents, Miami, Fla.) for sodium, potassium, and calcium using CDC serum standards. The precision of a single determination is better than 2% relative standard deviation for each element. Comparison with the certified values provided with the control serum indicates an accuracy of better than 2% for sodium and calcium, and 2.2% for potassium. The analysis time for the simultaneous determination of sodium, potassium, and calcium, including sample taking, analysis, and data acquisition is approximately two minutes. At present the data acquisition step--i.e., manually writing down the digital data for the lines and background-is the rate limiting step in the analysis. This time will be substantially reduced when a minicomputer is interfaced with the vidicon flame spectrometer to provide more rapid data acquisition. An important aspect of this method is the use of serum standards rather than aqueous synthetic standards. Use of aqueous standards results in erroneously high values for the determination of sodium, potassium, and calcium in control serum as shown in Table IV. A comparison of analytical curves for these elements using serum standards and aqueous standards obtained using identical flame conditions is shown in Figure 5. The normal physiological range (8) is also shown on these curves for comparison. (8) P. B Hawk, 8. L. Oser, and W. H. Surnrnerson. "Practical Physiological Chemistry," 13th ed., McGraw-Hili Book Co., New York, N.Y., 1954, p 499.
ANALYTICAL CHEMISTRY, VOL. 46, NO. 9 , AUGUST 1974
29
::I 40
37
o
t
,
C
/
// 16
I5 14
5
COnCenlratlOn, meq I P
6
7
8 9 IO I1 I 2 I3 Concenfrotion, m g / d l
I4
I5
Figure 5. Calibration curves (a) Sodium: (b)Potassium: (c) Calcium. (0)CDC serum standards. (A)aqueous standards
The increase in signal using serum is probably due not only to changes in flame conditions caused by the introduction of proteins into the flame, but also to changes in the solution nebulization caused by differences in surface tension and viscosity. A similar finding for calcium was observed by Lott and Herman (9).Attempts to overcome this effect by variation of the fuel-to-oxidant ratio and flame observation height were unsuccessful. T o evaluate this method under actual clinical conditions, human serum samples from hospital patients were analyzed. Table V shows the results of the simultaneous determination of sodium, potassium, and calcium in ten serum samples using the vidicon flame spectrometer. Actual values for the identical serum samples obtained by the clinical laboratory a t the hospital are shown for comparison. For sodium and potassium, they employed an Instrumentation Laboratory Model 143 flame photometer. This instrument automatically dilutes the serum samples (1:200) using a peristaltic continuous-flow dilutor. A lithium internal standard is automatically added to the samples when they are diluted. Since the instrument is a continuous-flow device, the amount of serum consumed depends on how long the sample is aspirated. Sodium and potassium were determined simultaneously with this system using an air-propane flame. The hospital values for calcium were obtained using the cresolphthalein complexone colorimetric method ( I O ) . This method requires 100 pl of sample and a ten-minUte incubation time. 8-Hydroxyquinoline is used to retard the magnesium interference. Potassium. The standard deviation of a single determination of potassium was estimated from five successive injections of control serum to be 0.05 mequiv/liter using the vidicon flame spectrometer. This gives a relative standard deviation of 1.19% for the determination of potassium, which compares well with the reported standard deviation (11) for the hospital system of 1.2 to 2.0% for potassium. The average deviation of the vidicon flame spectrometer values from the clinically obtained values shown in Table V is 3.22%. A standard t-test for paired variance indicates that a t the 95% confidence level, the two sets of data are equivalent. As shown in Table IV, the vidicon flame method has an accuracy of 2.2% for the determination of potassium in a serum standard. Sodium. The standard deviation of a single determination of sodium was estimated from five successive injections of control serum to be 0.50 mequiv/liter using the vid(9) J A. Lott and T. S. Herman, Clin. Chern., 17, 614 (1971). (10) M. E. Chilcote and R. D. Wasson. Clin. Chern., 4, 200 (1958).
Table V. Analysis of Hospital Serum Samples ~
_
Vidicon
Patient
1 2 3 4 5 6 7 8 9 10
Sodium, mequiv, 1.
143 139 140 144 145 131 141 142 142 145
_
Potassium,
_ mequiv,'I.
Calcium, mg, dl
Clinical Vidicon Clinical Vidicon Iieferee
142 137 136 143 145 134 144 142 142 143
3.80 4 .OO 4.45 4.02 4.25 4.33 3.93 4.46 4.06 3.97
3.7 4.1 4.6 4 .O 4.4 4.6 4.1 4.8 4.1 3.9
9.56 9.69 9.83 9.44 9.67 8.07 9.67 9.79 9.58 9.76
9.37 9.76 10.4 9.23 9.75 8.06 9.48 9.47 9.65 9.81
Clinical
9.4 9.6 9.6 9.1 9.6 8.0 9.2 9.6 8.9 9.2
icon flame spectrometer. This gives a relative standard deviation of 0.36% for the determination of sodium using this method which compares well with the reported standard deviation ( 1 1 ) for the hospital system of 0.9% for sodium. The average deviation of the vidicon flame spectrometer values from the clinically obtained values shown in Table V is 1.18%.A standard t-test for paired variance indicates that, even for a confidence level of only 55%,there is no significant difference between the two sets of data. As shown in Table IV, the vidicon flame method has an accuracy of 1.4%for the determination of sodium in a serum standard. Calcium. The standard deviation of a single determination of calcium was estimated from five successive injections of control serum to be 0.17 mg/100 ml using the vidicon flame spectrometer. This gives a relative standard deviation of 1.75% for the determination of calcium using this method. The average deviation of the vidicon flame spectrometer values from the clinically obtained values is 3.12%. A standard t-test for paired variance indicates that, a t a confidence level of greater than 99%, there is a significant difference between the two sets of data. For this reason, the calcium determinations were also run on the same samples using an optimized single-element flame emission procedure. A 50-11 sample of each serum was added to a 10-ml volumetric flask containing 0.4 ml of butanol as an antifoaming agent, and diluted to volume. Samples of the three CDC serum standards were diluted in the same man( 11) R J. Schlestnger. R A. Lesonsky, and R Lottritz, Clin. Chern., 18, 1005
(1972). A N A L Y T I C A L CHEMISTRY, VOL. 46. NO. 9 , A U G U S T 1 9 7 4
1235
ner. A calibration curve was made by aspirating the diluted CDC serum standards into the nitrous oxide-acetylene flame in the conventional manner. The calcium 422.7-nm line was determined using the vidicon flame spectrometer with a slit width of 20 Fm. No filters were employed. The results of these determinations are listed in Table V as referee values for calcium. The average deviation between the simultaneous multielement vidicon results and the singleelement flame emission calcium determinations is 1.81%.A standard t-test for paired variance indicates that even for a confidence level of only lo%, no significant difference exists between these sets of data. Considering the good agreement obtained in the analysis of control serum shown in Table IV, and the good agreement obtained with the referee method, the vidicon results are considered to be more reliable than the calcium determinations made using the cresolphthalein complexone method. CONCLUSIONS
The success of the vidicon flame spectrometer for the simultaneous determination of sodium, potassium, and calcium in serum results from a combination of instrumental components and processes. They act in a complementary fashion, such that elimination of any one obviates the goals achieved. The nitrous oxide-acetylene flame is necessary to eliminate the phosphate interference on calcium and to provide the necessary excitation for the detection of the chosen potassium lines with the vidicon. The sample introduction system allows the direct injection of small samples into the flame. The vidicon detector allows simultaneous measurement of intensities over a range of wavelengths and also permits integration of the transient signals produced by the sample introduction system. The wide spectral response of the detector permits the detection of the long wavelength sodium lines. The analytical lines chosen permit the direct determination of sodium, potassium, and calcium at the levels encountered in serum without dilution, and the chosen window allows the monitoring of these lines in an apparent 40-nm window which avoids detector
saturation from flame bands and other intense analyte emission lines. The advantages of the vidicon flame spectrometer are speed, simplicity, ease of operation, accuracy, precision, and versatility. Compared with most flame systems used in clinical analyses, which generally determine only two elements simultaneously, the vidicon flame spectrometer permits the rapid simultaneous determination of the three most frequently clinically determined cation electrolytes on a single sample. At present, the system is operated in a manual mode. The system, however, is easily amenable to automation, and future efforts in this direction will substantially reduce the analysis time still further. The system is not only simple to operate, but possesses no mechanical moving parts, and should therefore be reasonably maintenance-free. Since the analysis is direct and requires no sample manipulation, potential sources of error are eliminated. Samples can be analyzed with an accuracy of 2.2% or better with a precision of 2% or better. The system is versatile and may be used for the determination of other cation electrolytes such as magnesium and lithium as well as other trace elements present in serum. The system may also be applied to the analysis of other body fluids, such as urine, by using appropriate standards. ACKNOWLEDGMENT
The authors would like to thank J. H. Boutwell and D. D. Bayse of the Center for Disease Control, Atlanta, Ga., for supplying the samples of analyzed bovine serum. Thanks are also extended to the National Bureau of Standards for supplying the results of the isotope dilution mass spectrometric analyses of the CDC samples for potassium. Finally, thanks are extended to Robert McGovern of the Tompkins County Hospital for providing the human serum samples and analyses. Received for review March 4, 1974. Accepted April 15, 1974. This work was supported by the National Institutes of Health, Grant No. GM-19905-02.
Hollow Cathode Ionization for the Mass Spectrometric Analysis of Conducting Solids B. N. Colby'
and C. A. Evans, Jr.
Materials Research Laboratory, University of Illinois, Urbana, 111. 6 180 1
A simple hollow cathode ion source for the mass spectrornetry of conducting solids is described. Using clean argon for the discharge support gas, broad elemental coverage was obtained with detection limits in the sub-ppma region possible. The presence of molecular gases in the discharge del Present address, E. I. du Pont de Nemours and Co., Inc., Instrument Products Division, 1500 S. Shamrock Ave., Monrovia, Calif. 91016.
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ANALYTICAL CHEMISTRY, VOL. 46,
NO. 9,
creased sensitivity by as much as three orders of magnitude. Ions representative of the hollow cathode sample appear primarily as singly charged, monoatornic species although some polyatornic ions were observed. Ionization of the elements is such that standardless analysis to within a factor of three is possible. Highest sensitivity is achieved using a high current, high voltage discharge, although this may result i some nonuniform ionization for volatile elements.
AUGUST 1974
