1849
V O L U M E 2 7 , N O . 11, N O V E M B E R 1 9 5 5 Modified Flame Photometer for Microdetermination of Sodium and Potassium A. K. Solomon and David C. Caton, Biophysical Laboratory, Harvard Medical School, Boston 15, Mass.
T IS
necessary to determine very small quantities of sodium
I and potassium in connection with studies on the perfusion of
single kidney tubules in the Secturus in vivo, which are currently under way in this laboratory. The collected fluid after perfusion amounts to 1 p1. or less, and contains, as a maximum, 95 millimicromoles of sodium and 2.8 millimicromoles of potassium. These quantities are too small to be measurable in normal commercial flame photometers. Consequently, a new detector for the Beckman flame photometer (Model DU) has been designed to make the necessary microdetermination of sodium and potassium. The availability of the RC.4 Type 6217 photomultiplier tube has made it possible to measure both elements with a single detector, using the most favorable flame emission lines, 589 mp for sodium and 768 mp for potassium. The photomultiplier has an S-10 spectral response, high (approximately 76%) in the range of sodium flame emission, and lox (approximately 2oJ,) but still usable in the range of potassium emission. Unfortunately, the tube is available only with a 1.5-inch photocathode, much larger than is required for the present service, and the unused area of the photocathode contributes unwelcome noise to the signal.
tained, but all other parts wit'hin the box have been discarded. Although the shutter is not required in normal operation, its use makes it easy to segregate tube noise from flame noise when finding the optimal conditions of operation. The box is extended by a brass cylinder which completely encloses the photomultiplier tube, socket, and chain of resist'ors, making the entire assembly insensitive to external light and signal interference. The circuit diagram is shown in Figure 2. All resistors are precision, although there is no evidence that this precaution is essential. As the G terminal of the electrometer is not a t ground potential, the positive lead (ground) of the high voltage supply must not be grounded to the rest of the system and a 1 to 1 isolation transformer is desirable in the 110-volt input of the high voltage supply. The Brown recorder should be grounded to the electrometer-monochromator ground, which should be connected to a good building ground. Care must be exercised in obtaining a good light seal, as the modified instrument is very sensitive to extraneous light. Because of this, it was found advisable to run an additional black painted brass tube inside the instrument from the output of themonochromator to the shutter. The tube is machined to fit on one end int.0 the recess surrounding the exit port of the monochromator, and on the other end into the recess around the shutter opening. All exposed inside parts are painted black, and all outside apertures are covered with black Scotch e1ectric.d tape. A smaller section of brass tubing fastened onto the end of the large brass phototube enclosure shields the anode-output conductor. This smaller cylinder and central conductor are fashioned to allow direct mounting of the electrometer preamplifier head, thus providing a minimum signal loss and maximum shielding to the electrometer input.
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DESCRIPTION OF APPARATUS
250 K i 1 % 0 5 W NA-I5
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Figure 1 shows a block diagram of the complete equipment.
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TERMINAL
GND "4 POWER SUPPLY ONLY t
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7. 40 MEG
INPUT OF VlBR4TlNG REED ELECTROMETER
A Beckman DC spectrophotometer \?ith Model 9200 flame attachment is used t o provide the signal. The box hich normally contains the phototubes and amplifier in the spectrophotometer has been modified to hold the 6217 tube. The high voltage for the tube is supplied by a Xuclear Instrument and Chemical Corp. Model 1090 power supply. As the phototube voltage should never normally exceed 800 volts because of phototube noise limitations, a smaller pori-er supply may be used, provided it has equally good regulation and stability characteristics. The output of the photomultiplier, a voltage drop across R 40-megohm 1-ictoreen resistor, is impressed across the input terminals of an iipplied Physics Corp. hlodel 30 vibrating reed electrometer. The electrometer in turn feeds a Brown 10-mv., h e c o n d , recording potentiometer 1% hich provides a w i t t e n record of the flame output. -500
REDUCING VALVES
a
BECKMAN
GAGES
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,,-6217
F L A M E PHOTOMETER
I
SUPPLY
Circuit diagram for 6217 tube
PHOTOTUBE
,
HIGH VOLTAGE
Figure
Figure 2.
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VIBRATING ELECTROMETER
ELECTROMETER
BROWN RECOROER
1. Block diagram of flame photometer assembly
The oxyacetylene flame attachment of the Beckman operates in the normal fashion, except for regulation of the gas supply. The regulation valves built into the flame control panel are kept constantly open, and the pressure is controlled instead by Kendall precision regulators (Model 10A). As these valves normally obtain their good regulation by bleeding a small fraction of the input gas to the ambient atmosphere, i t is necessary to specify that the acetylene valve be modified to omit this feature. The modifications to the Beckman amplifier box t o enable it to hold the 621T are straightforward. The shutter has been re-
The vibrating reed electrometer provides absolutely stable amplification of the moderately high impedance signal. The Brown recorder is an essential part of the system, as a written record is necessary for the measurement of samples of 0.5 ml. or less. Samples of 0.5 ml. can be measured satisfactorily using the small beakerlets supplied with the flame photometer. Sma!ler samples require the use of special cups which can be machined from Lucite to fit the specific needs of the problem. The sample holder can be adjusted so that the aspirator can suck the Lurite cups almost entirely dry. RESULTS
The performance of the modified flame photometer is conipletely satisfactory for sodium. The standard deviation of R set of 10 determinations on 1.0-ml. samples of 100 micromolw of sodium per liter (100 millimicromoles of sodium per sample) is 0.6%, when the same cup is used for all samples (instrumeiit
ANALYTICAL CHEMISTRY
1850 settings: slit width, 0.01 mm.; phototube, 620 volts; and electrometer sensitivity, 100 mv.). The standard deviation of a set of 10 determinations on 0.5-ml. samples of 1 micromole of sodium per liter (500 niicronicromoles of sodium per sample) is 1.4%. In practice, the limit of detectability seems to be set by the sodium contamination from deionized water and glass containers.
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DEI3NIZED W A T E Q FLAME
06 FLAME J
are equal to concentrations of 8.7 millimicromoles of sodium and 25.6 millimicromoles of potassium per liter. The manufacturer further states that the detection limit is generally equivalent to the error in concentration measurement, and that the detection “limits indicated may be reached only under optimum Conditions.” From these figures it may be calculated that the Model DU with photomultiplier attachment will be able to measure concentrations of 0.6 micromole of sodium per liter to an accuracy within &1.4% and 0.3 micromole of potassium to an accuracy within &8%. These calculations may be compared with the accuracy obtained in the present study: for sodium, Concentrations of 1 micromole per liter have been measured to 3 ~ 1 . 4 %and for potassium, concentrations of 0.6 micromole per liter have been measured to i870.The authors’ results are slightly less accurate than the values calculated from the manufacturer’s optimum performance data.
Figure 3. Tracing of Brown recorder of signal from potassium chloride solutions
3.5
R a t e of consumption of fluid is 1.1 ml. per scale divisjon, a n d the record mo”es a t SO seconds per scale dirision. The notation flame” refers t o the sieiial when no solution is being aspirated into the burn& a n d t h e base line is taken as the signal when water purified by passage through a n ion exchange column (Barnstead Bantam derninernlizer) is aspirated
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I t is more difficult to obtain satisfactory microdeterminations of potassium than sodium. The potassium concentration i.1 the solutions under study is much smaller than that of sodium, and the photomultiplier response is very poor at 768 mp. Whereas tube noise and flame noise are effectively absent in the sodium measurements, each contributes significantly to the error in the potassium measurement. Although it is possible to get a much larger signal lvhen the vibrating reed electrometer is set a t the 10-mv. sensitivity for the potassium determinations, the records appear to be more easily measurable \Then the electrometer is set at 100-mv. sensitivity. Figure 3 shows a tracing of the record obtained on samples of 0.5 ml. or less of solutions containing graded amounts of potassium running from 0.6 up to 10 micromoles per liter. These values are plotted against potassium concentration in Figure 4 (instrument conditions: slit width, 0.24 mm.; phototube, 720 volts; and electrometer sensitivity, 100 mv.). The standard deviation of sets of 10 duplicate samples measured under these conditions ranges from 1.5y0 for 6 micromoles per liter up to 8% for samples containing 0.6 micromole per liter. As these samples contained about 0.3 ml. of solution, the smallest sample represents a measurement of 200 micromicromoles of potassixm. Under the experimental conditions, there is no interference effect when potassium chloride in concentrations of 0 to 16 micromoles per liter is added to sodium chloride at a concentration of 100 micromoles per liter. There is a small increase in signal when sodium chloride in concentrations of 50 to 100 micromoles per liter is added to potassium chloride in concentrations of 2 to 4 micromoles per liter. At a concentration of 4 micromoles of potassium and 100 micromoles of sodium per liter, the increase amounts to 0 . 0 6 7 ~ / ( r n i c r o m o l of e ~sodium per liter), so that changes in sodium concentration of 10 micromoles per liter or less may be neglected in this concentration range. The results obtained with the modified flame photometer may be compared with the manufacturer’s specifications for the Model DU photometer with photoInultiplier attachment. The detection limits are given as 0.0002 p.p.m. for sodium a t 589-mp wave length, and 0.001 p p m . for potassium a t 766.5-mp [Beckman Instruments, Instruction Manual No. 334, June 1954: Gilbert, P. T., Jr., Ind. Labs., 3, 41 (August 1952)]. These
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4
6
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CONC.OF K IN p M / L
Figure 4. Response of flame photometer to potassium chloride solutions of graded concentrations Points taken from record shown in Figure 3
However, the present modification was designed to measure very small quantities of sodium and potassium. The quantity of solution used to obtain the results given above comprised 0.5 ml. for sodium and 0.3 ml. for potassium. In practice, samples of 0.25 ml. are routinely used with no apparent sacrifice in accuracy. These volumes may be compared with the capacity of the standard beaker used in the photometer with photomultiplier attachment, lvhich contains 5 ml. of solution. Thus, the modified spectrophotometer makes it possible to measure samples of one tenth or less of the normal volume with an accuracy comparable to the best that can be obtained with samples of normal volume under optimum conditions. ACKNOWLEDGMENT
The authors express their thanks to Joseph C. Shipp for carrying out the replication measurements. This work has been supported in part by the Atomic Energy Commission.
