Measurement of Picomole Amounts of Carbon Dioxide by Calorimetry G. G. Vurek Laboratory of Technical Development, National Heart and Lung Institute, Room 5D- 18, Building 10, Bethesda, Md. 200 14
D. G. Warnock Laboratory of Kidney and Electrolyte Metabolism, National Heart and Lung Institute, Bethesda, Md. 200 14
Roland Corsey Electrical Engineering Section, Biomedical Engineering and Instrumentation Branch, Division of Research Services, National Institutes of Health, Bethesda, Md. 20014
The measurement of picomole amounts of bicarbonate and carbon dioxide is useful in the studying of the mechanisms by which the kidney controls body pH. These studies may involve collection of a few nanoliters of fluid containing less than 300 picomoles of carbon dioxide (as bicarbonate ion and dissolved gas) ( I ) . Small amounts of carbon dioxide have been measured indirectly with pH electrodes (2-4), conductimetrically (5, 6), by conversion to methane with subsequent analysis using a flame ionization detector (7), and by measuring the pressure rise when carbon dioxide-containing samples are mixed with acid in a closed system (8). Mass spectrometers can also be used. The heat of reaction of carbon dioxide with lithium hydroxide has been proposed as an atmospheric CO2 monitoring scheme (9). This paper describes a simple apparatus to measure the heat released when LiOH and COZ react and its application to the measurement of carbon dioxide with a sensitivity of less than 10 picomoles. The overall reaction between COz and LiOH, 2 LiOH CO2 LiZC03 + H20; AH = -8.96 X 104J/mole COS, is made up to two reactions (10):2 LiOH 2H20 s 2 LiOH H20; AH = -1.214 X 105J/2 moles HZO;and 2 LiOH H20 COZ Li2CO3 3H20; AH = +3.18 X 104J/mole COz. If the LiOH is maintained in the dehydrated state, there is a net exothermic reaction and the water released by the formation of LiZCO3 can activate other LiOH molecules so that the overall reaction is self-sustaining. A dose of CO2 will produce a transient rise in the temperature of the LiOH; both the peak value and the integral of the temperature change are proportional to the amount of COz. We use a base-line-correcting integrator which minimizes the effect of short-term noise.
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EXPERIMENTAL Figure l a shows the block diagram of the analytical system. Dichlorodifluoromethane carrier gas a t constant pressure passes through a carbon dioxide scrubber and a Mg(C104)2 drying column to a pair of capillary flow restrictors which provide a constant flow of carrier gas. Flow restrictors were made from lengths of 3.9 X 10-"-cm i.d. glass capillary; the length was selected on the basis of the equation for viscous flow (11) to give the desired flows a t reasonable pressures. Each capillary was sealed with silicone rubber adhesive into a piece of stainless steel tubing to give the flow restrictor strength. One stream washes C 0 2 released from the acid chamber to a T tube where it joins with the other stream to dilute any residual water vapor below the equilibrium value for LiOH . H20. The release chamber is made of 2.5-mm 0.d. quartz tubing, with side arms for the carrier gas and a small bulb for a mercury seal. The side arms are about 1.3-mm 0.d. and the overall length is 3 cm. Samples are injected with a nanoliter-volume pipet, the outside diameter of which is slightly smaller than the constrictions of the bulb. A halocarbon film coats the inner surface of the release
chamber to prevent the concentrated HsP04 from creeping out of the bottom well. Heavy walled 0.015-inch i.d. vinyl tubing of the sort used in AutoAnalyzer pumps is used to connect the various components. A single granule of LiOH, weighing about 0.6 mg, is wedged between the two thermistors which form the active arms of the temperature sensor bridge and is held in place with a small amount of grease. The thermistors are mounted in a 7-mm diameter X 2-mm deep chamber with a removable cap (not shown in the figure). The chamber is mounted on the same box as the preamplifier indicated in Figure l b and the whole is surrounded by 1 cm of polystyrene foam. The electronics circuit (Figure l b ) consists of a simple lOOOX bridge amplifier, a post-amplifier with a gain of 5, and a base-linecorrecting integrator. Each sample of COz produces a transient change in the temperature of the LiOH granule and the integral of the resulting bridge unbalance signal is proportional to the amount of carbon dioxide in the sample. The voltage-to-frequency converter (V-F) provides pulses a t a frequency proportional to the signal from the amplifier. We start a measurement cycle by closing a switch. The signal is integrated (counted) with a minus sign for T/20 min; then a tone signals the operator to inject the sample. The signal, divided by 10, is integrated with positive polarity for T min. Finally, the input is again integrated with a minus sign for T/20 min, and the accumulated count is the base-line-corrected integral. The integrator uses the mains frequency as the time-base and i t s digital design eliminates internal drift. The overall cycle time is 2.2 minutes. Materials. The flake thermistors, 1 mm square, 50 microns thick, 2 megohms f 20%, slope matched f 0.5% at 25 C are available from Thermornetrics, Inc., 15 Jean Place, Edison, N.J. 08817, Cat. No. 2F40 P205/S13A, or as a set of two matched pairs mounted on a transistor-type header, Cat. No. 2F40/80/M205/547. Granular lithium hydroxide was obtained as Lithasorb (trade mark of Foote Mineral Co.), Fisher Scientific, Cat. No. L-177. Dichlorodifluoromethane was obtained from a 426-g can of refrigerant grade material. A type 70 low pressure regulator (Matheson Gas Products) controlled the pressure of the system. Phosphoric Acid: 8596, reagent grade, 14.8M. The complete diagram of the integrator is available as drawing number 073-1695, Electrical Engineering Section, Biomedical Engineering and Instrumentation Branch, Division of Research Services, National Institutes of Health, Bethesda, Md. 20014. The quartz chamber is coated with FC-709, 3M Company, Chemical Division, Minneapolis, Minn. 55119. Procedure. Samples and NaHC03 standards are injected through the mercury seal into the acid drop. Because of the small volume of the samples, usually 10-15 nl, the loss of dissolved carbon dioxide can be a problem and samples of kidney tubule perfusate are analyzed immediately after collection. The phosphoric acid must he changed after about 30 analyses to keep the acid from overflowing into the gas path. Granules are changed every two weeks or so.
RESULTS AND DISCUSSION The instrument was tested for linearity of response, precision, and sensitivity to potential interfering substances. Solutions of NaHC03 with concentrations between 0 and A N A L Y T I C A L C H E M I S T R Y , VOL. 47,
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Figure l a . Block diagram of the COP analyzer The flow resistance of the capillary flow restrictors, given in SI units, was measured at 4 X
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Figure lb. Block diagram of the electronic circuitry of the COP analyzer The preamplifier is a National Semiconductor, Inc., device. The “floating” bridge source is made of 2 TR113R batteries. The actual value of the thermistors available may require a slightly different balancing circuit
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25mM were used for tests of linearity and precision. Typical results gave a linear regression, correlation coefficient greater than 0.99, and a standard deviation of 0.5mM corresponding to 6.5 picomoles with a 13-nl pipet (Figure 2). Samples and standards are interspersed to obtain good accuracy and to correct for slow changes in sensitivity. The sensitivity of individual lithium hydroxide granules varies with their mass so that each gives a slightly different calibration curve but each stays constant within f 2 0 % over its useful life, which may exceed two weeks. We normally obtained about 2 integrator units per picomole. 766
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Carbon dioxide is the principal acidic gas to be released by the phosphoric acid but the samples may contain both acetate and lactate ions which could interfere, as the vapor pressure of their acids is reasonably high a t room temperature. We found no interference by these materials at levels below 5 mM. Water vapor is involved in the overall reaction of LiOH with C02. We store the LiOH over silica gel and use a dry carrier gas to maintain exothermic reaction conditions. Although the presence of some LiOH HzO is necessary for reaction with C02 (12), only a small amount is needed and the system retains its responsiveness for as long as we can keep the LiOH dry. We have noted a transient exothermic response to water at high (4 jd/sec or more) carrier flow. We use carrier flows below this value so that the water “blank” is small and adds little to the error of measurement. The response may be due to a transient release of water vapor which may result from the heat of dilution of the acid. A t room temperature, the vapor pressure of the acid is well below the equilibrium pressure for LiOH HzO. At the typical carrier flow of 2 wl/sec, the signal reaches a maximum value 30 seconds following injection and returns to base line within 2 minutes. The peak output signal is about 150 fiV for a 200-picomole sample. Short-term amplifier noise (0.1-1 Hz) is about 2 FV peak-to-peak referred to input. In order to achieve maximum sensitivity, we made the thermal mass and conductance as low as possible. On the basis of experiments done with two chambers in series, we feel that at least 80% of the COz in our samples reacts with the LiOH. Sensitivity could be improved by reducing the volume of the chamber and using a lock-in amplifier to eliminate the l / f noise which appears in conventional dc amplifiers. Additional protection for ambient temperature changes may also be needed as the noise is equivalent to 10 MK peak-to-peak. Sensitivity and precision are adequate
for our purpose, and we can analyze more than 20 samples and standards per hour.
ACKNOWLEDGMENT The authors thank R. L. Berger, NHLI, and E. J. Prosen, NBS, for their advice on calorimetry and the thermochemistry of the reaction of LiOH with C02.
LITERATURE CITED (1) M. Burg and N. Green, Kidney Int., 4, 301 (1973). (2) C. R. Caflisch and N. W. Carter, Anal. Biochem., 60, 252 (1974). (3) B. Karlmark and M. Sohtell, Anal. Biochem., 53, 1 (1973).
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(6) F. J. A. Prop, f x p . Cell Res., 7,303 (1954). (7) F. W. Williams, F. J. Woods, and M. E. Umstead. J. Chromatogr. Sci., 10, 570 (1972). (8) F. Hevert, Pflugers Arch., 344, 271 (1973). (9) w. J. Jones, US. Patent 3716337 (1973). (10) D. A. Borfla and A. J. Mass, hd. Eng. Chem., Process Des. Dev., IO, 489 (1971). (1 1) S. Dushman, "Scientific Foundations of Vacuum Technique", John Wiley and Sons,New 'fork, N.Y.. 1949, p 84. (12) D. D. Williams and R. R . Miller, lnd. f n g . Chem., fundam., 9, 454 (1970).
RECEIVEDfor review September 6, 1974. Accepted December 19,1974.
Convenient Preparation of Standard Thiosulfate Solutions Edwin H. Funk and Albert W. Herlinger Department of Chemistry, Loyola University of Chicago, Chicago, IL 60626
The preparation of standard thiosulfate solutions of sufficiently high concentration to be of practical utility in iodometric titrations has relied almost exclusively upon sodium thiosulfate pentahydrate. Although standard thiosulfate solutions may be prepared by direct weighing of Na2S203.5H20 or the anhydrous sodium salt (1,2),special reagents and care are necessary to ensure a definite composition of these materials. Additionally, solutions of sodium thiosulfate slowly change in titer with time and require periodic restandardization. In actual practice, approximate solutions using ordinary reagents are prepared and standardized against primary standard materials. To obviate the problems associated with Na2S203 solutions, we initiated a search for a primary standard thiosulfate salt which would exhibit good stability characteristics. A convenient method for preparing standard thiosulfate solutions utilizing barium thiosulfate monohydrate as a primary standard has been devised. Plimpton and Chorley, as early as 1895, suggested the use of this salt as a primary iodometric standard ( 3 ) . However, it was not until 1953 that there were any quantitative data ( 4 ) to support this suggestion. Barium thiosulfate monohydrate, which may be obtained commercially from several sources, has a high equivalent weight (267.51 amu) and can be dried to constant weight without special procedure at 40 "C. Above about 50 OC, the hydrate slowly loses water, and the effects 'of high drying temperatures have been reported previously ( 4 ) . The solubility of the salt, 0.283 gram of Bas203 per 100 cm3 of saturated solution at 25 "C ( 5 ) , is insufficient to allow preparation of a 0.1Nsolution. Consequently, its application has been restricted almost exclusively for use as a solid in the standardization of 12 solutions. We have utilized the complexing ability of EDTA, disodium (ethylenedinitri1o)tetraacetic acid dihydrate, to increase the solubility of BaS203. H20. The conditional stability constant for the EDTA-Ba2+ complex (log K = 6.4) a t pH 9 (6), the optimum pH region for stabilizing thiosulfate solutions, indicates that the EDTA concentration should be approximately 0.1F if the desired concentration of S 2 0 3 2 - is to be achieved. Thiosulfate solutions prepared by direct weighing of BaS203. H20 and addition of EDTA are shown to be suitable for several applications of the iodometric method; however, the presence of EDTA may interfere in determinations in which the redox potential or reversibility of the
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reaction are adversely affected. The barium thiosulfate solutions appear to have the additional advantage of stability characteristics which are superior to the more conventionally prepared thiosulfate solutions.
EXPERIMENTAL Apparatus. The p H of the solutions was determined with a Corning Model 12 research p H meter. Reagents. Primary standard grade barium thiosulfate monohydrate was purchased from Sargent-Welch Scientific and was used without further purification. Primary standard grade KI03, KzCrz07, and copper were used. Hydrogen peroxide was 3?6 Fisher certified reagent grade Hz02 and commercial Clorox was used as an unknown hypochlorite solution. The EDTA and all other chemicals were analytical reagent grade materials and were used without further purification. Procedure. Finely ground BaSz03. HzO was dried at 40 "C for 2 hours and weighed exactly to prepare 0 . 1 N thiosulfate solutions (26.751 g k ) . Two methods were used in preparing the standard thiosulfate solutions with equally good results. In both cases, triple distilled water which had been recently boiled was used. Method A . An amount of EDTA (37.224 g/L) equivalent to the previously weighed Bas203 H20 (26.751 g/l.) was dissolved in exactly twice the equivalent amount of 1 N NaOH (188 ml of 1.065N base). This solution was used to dissolve the BaS203. HzO which had been placed in a 400-ml beaker. The resulting mixture was stirred with a magnetic stirrer to facilitate rapid and complete dissolution and then transferred quantitatively to a volumetric flask. After diluting to the mark and mixing thoroughly, the solution exhibited a p H of 9.5. Method B. The procedure outlined in Method A was followed except that the requisite amounts of EDTA and NaOH calculated from the weight of BaS203. H z 0 to be dissolved were weighed to within 0.1 gram and added to 150 ml of H20. Solutions prepared in this manner have a p H in the range of 8 to 10 and are as applicable as the more carefully prepared solutions in Method A. Large excess of base, i.e., 2 or 3 grams more than that necessary to neutralize the EDTA, causes the titer of the solution to be high and this is to be avoided. Standardization, Stability, and Applicability of Bas203 Solutions. Standardization of the thiosulfate solutions was accomplished by titrating vs. potassium iodate using the method described by Kolthoff (7). These solutions were then restandardized at approximately weekly intervals and found to be stable for a t least six weeks when excess base was avoided. The applicability of the Bas203 solutions was tested in the iodometric determinations of dichromate, iodate, hydrogen peroxide, hypochlorite, and copper using standard procedures (8). Generally, acidifications of the solutions to be titrated were accomplished using hydrochloric acid in preference to sulfuric acid since it was felt that the precipitation A N A L Y T I C A L C H E M I S T R Y . VOL. 4 7 , N O . 4 , APRIL 1 9 7 5
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