Anal. Chem. 1988, 60, 2413-2418
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Determination of Sodium and Potassium in Nanoliter Volumes of Biological Fluids by Furnace Atomic Absorption Spectrometry Lori A. Nash Faculty of Health Sciences, Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada
Linda N. Peterson Faculty of Health Sciences, Departments of Pediatrics and Physiology, University of Ottawa, Ottawa, Ontario, Canada
Steven P. Nadler Faculty of Health Sciences, Department of Medicine, University of Ottawa and Ottawa General Hospital, Ottawa, Ontario, Canada
David Z. Levine* Faculty of Health Sciences, Department of Medicine. University of Ottawa and Ottawa General Hospital, Ottawa, Ontario, Canada KlH 8M5
Renal tubular fluid samples are nanoiiter (lo-' L) volumes containing sodium and potassium concentrations that are within the range of determinatlon by furnace atomic absorp tion. Modlficatkn of nandlter handHng techniques and the use of microboats with the I L 951/655 provided a method for rapid precise analyses (relative standard deviation of 5 % ). Determinatlons of sodium and potassium were precise; however, Inaccuracies occurred with anion substitution of sodium salts. NaHCOssoMions gave conslstentiy higher peak height absorbance and area absorbance compared with those of NaCk the peak area absorbance correlated linearly with the concentration of bicarbonate. Pretreatment of the microboat with boric acid eiimlnated this phenomenon and the assoclated inaccuracy. Comparison of determination of sodium In nanoffler samples by graphite furnace atomic absorption with macroanalysis by flame emisslon gave relative errors of less than 2.0%. Additkn of sodium and potassium to tubular fluid samples yielded mean recoveries of 102.6% and 99.7 %, respectively. We conclude that graphite furnace can be an accurate method for measurement of sodium and potassium in nanoiiter volumes of biological fluids.
Determination of elements in ultramicrovolumes or quantities of biological samples has been the subject of intense interest during the past 20 years (1-6), particularly in the field of renal physiology. Micropuncture and microperfusion techniques have provided the renal physiologist access to various segments of the functioning nephron of the kidney. Measurement of changes in tubular fluid electrolyte concentrations in these segments contribute to our understanding of the mechanism of urine formation. Although renal tubular fluid samples contain elements in concentrations exceeding the usual analytical range of the graphite furnace, the nanoliter volumes collected by micropuncture create unique problems for analysis. Kuntziger et al. (1)introduced an original method for the determination of calcium in nanoliter volumes of renal tubular fluid by furnace atomic absorption. Good and Wright (2) used a similar approach for the determination of sodium and potassium in renal tubular fluid. However, details of the nanoliter sample handling techniques and instrument pa0003-2700/88/0360-2413$01.50/0
rameters have not been published. Further, little is known about anion interferences. The objectives of this paper are (1) to describe the ultramicrosample-handling techniques used in our laboratory for the determination of sodium and potassium in 0.1-nL aliquots of renal tubular fluid, (2) to demonstrate the accuracy of micro atomic absorption (AA) analysis by recovery studies and comparisons with flame emission (FE), and (3) to study anion' substitution and its effect on atomization profiles of sodium and potassium, with particular attention to the enhancement of sodium absorbance by the HC03 anion, a solute normally present in tubular fluid.
EXPERIMENTAL SECTION Reagents. All reagents used were of Baker Analyzed grade with the exception of hydrochloric and nitric acid, which were of Ultrex quality. Sodium and potassium standards were prepared by dissolving Ultrex sodium and potassium chlorides in deionized distilled water. Concentrations of Na and K in our studies are expressed as mM. Standards and artificial tubular fluid quality controls were prepared in 100-mL volumes. Ten-microliter aliquots were analyzed by flame emission using the Instrumentation Laboratories (IL443) Flame Photometer. Concentrationsof sodium usually found in tubular fluid range between 10 and 150 mM or 23 and 345 pg for a 0.1-nL aliquot deposited on a microboat. Potassium in tubular fluid samples usually ranges from 1 to 100 mM or 3.9 to 390 pg for a 0.1-nL aliquot. The major constituentsof tubular fluid samples in these studies were Na, K, HC03, Cl, urea, SO4, mannitol, inulin, and lissamine green dye. Lissamine green is used to identify nephron segments in vivo. Artificial tubular fluid samples containing the solutes listed below were processed for analysis in a manner similar to that used for tubular fluid samples. These solutions contained the following: (A) 25 mM NaHC03, 65 mM NaC1,7.5 mM K&O4, approximately 0.1% lissamine green dye; (B) 25 mM NaHC03, 65 mM NaC1, approximately 0.1% lissamine green dye; (C) 25 mM NaHC03, 7.5 mM K2SO4, 102 mM urea, approximately 0.1% lissamine green dye; (D)140 mM NaCl, 5 mM KC1; (E) 120 mM NaCl, 2 mM KC1. Apparatus. Table I summarizes the instrument parameters used for the Instrumentation Laboratories Model 951 atomic absorption spectrophotometer and Model 655 controlled temperature furnace atomizer. Analysis was carried out simultaneously in two channels for Na and K. Analysis was carried out in double-beammode, the read-out mode was peak area, and the 0 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
Table I. Instrument Parameters for Analysis of Na and K in 0.1-nL Aliquots"
light source lamp current, mA wavelength, nm slit width, pm band pass, nm readout mode integration time, s purge gas flow rate, L/min indicated temp, "C time, s sample size, nL auto clean
sodium
potassium
HCL-IL63059
HCL-IL62863
8 589
8
320
320 1.0
1.0
A
766.5
peak area peak area 4 4 argon 5 75, 110, 750,850, 2000, 2000 5, 5, 10, 10, 0, 5 0.1-0.2 5 s, 2500 O C
Microboat with pyrolytic graphite rectangular cuvettes. atomization signal was integrated for 4 s for both Na and K. The controlled temperature furnace atomizer IL Model 655 was equipped with a pyrolytic graphite cuvette and a removable microboat or platform. The furnace was purged with argon at a flow of 5 L/min during the 5-s dry cycle, 1 L/min during pyrolysis, and 5 L/min after atomization. The graphic display of absorbance versus time during atomization was a critical tool in selection of a furnace temperaturetime program, as well as in discriminating poor sample analysis. A drifting zero line could be readily seen on the display. Procedures. Sample Handling. Tubular fluid samples and aqueous standards were collected in micropuncture pipets filled with Sudan-Black-stainedmineral oil and then capped with oil to prevent evaporation. These glass pipets are approximately 6 cm in length with a 6-pm fine bevelled tip. Each tubular fluid sample or standard was transferred to constant bore tubing for measurement of volume and divided into several aliquots for other analyses. Approximately 5 nL was placed in a micropuncture pipet and transferred to an oil-fiied (65 mm X 10 mm X 8 mm) quartz glass trough (Vitro Dynamics, Inc., Rockaway, NJ). The quartz trough was meticulously cleaned with Ultrex nitric acid, Ultrex hydrochloric acid, and deionized distilled water, siliconized, and fiied with high-viscosity mineral oil (Fisher 0-122 Saybolt Viscosity 335/365) before use. No signal for Na or K was produced when oil alone was applied to the microboat and treated as a sample. To facilitate sample transfer, samples and standards in micropuncture pipets were mounted in a metal pipet holder attached to a syringe via Tygon tubing. The pipet holder was fastened into a micromanipulator. The quartz trough was mounted on an adjustable bar connected to a micromanipulator. The trough and the pipet were viewed through a dissecting microscope. Approximately 5 nL of tubular fluid sample, standards, and 50 nL of water were deposited along a 2-cm length of the trough with sufficient distance apart to identify them. All aqueous samples were constantly covered with oil to prevent evaporation. Analysis was usually carried out within 3 h of sample collection. Delivery pipets for the transfer of 0.1-nL aliquots of sample to the microboat were hand-manufactured in our laboratory. Glass tubing approximately 6 cm in length was pulled over a flame to an internal diameter of approximately 0.1 mm and mounted into a vertical microforge equipped with a platinum heating loop and weights. With the aid of a stereomicroscope the tubing was melted and stretched to an internal diameter of about 25 pm, and two constrictions were made to isolate a length of approximately 250 pm. The constriction pipet was treated with halocarbon oil to prevent adsorption of aqueous sample to the exterior of the pipet and to improve delivery characteristics. The pipets were mounted into a larger glass tube and connected to an air displacement syringe via polyethylene tubing. The pipet bulb, which held approximately 0.1 nL, could not be seen without a microscope. The exact volume was determined by radioisotopic techniques, when required. A Bausch and Lomb stereo zoom microscope, Model KV1070, was adapted to hold a nanoliter delivery pipet rigidly fixed to a unique pipet mount. This pipet mount (Microanalytic Instrumentation, Bethesda, MD) provided three degrees of translational
B
Flgure 1. Nanoliter sample pipetting (70X magnification). Schematic diagram demonstrates nanolier sampling from quartz trough: A, 0.1-nL constriction pipet: B, aqueous samples.
freedom, so that the tip of the pipet could be positioned in the center of the field and the focus optimized for oil immersion. The microscope and pipet move on the same axis with the pipet always in focus. The quartz oil trough containing nanoliter samples was mounted on a micromanipulator bar such that the trough could be moved into the microscope's center of field. The microboat with the microboat grabber was mounted in a fixed position directly above the oil trough immediately prior to sample transfer and rapidly returned to the controlled temperature furnace atomizer after sample delivery. The 0.1-nL delivery pipet attached to the Bausch and Lomb microscope was lowered into the oil trough above the aqueous bubble, as seen in Figure 1. First, 0.1 nL of oil was aspirated up to the constriction. Next, 0.1 nL of aqueous sample was aspirated, and then the sample was capped with oil. The microscope was raised and moved directly above the microboat. The pipet was carefully lowered onto the surface of the microboat, and the oil cap and entire aqueous sample were delivered rapidly as a bolus. The microboat's immediate transfer to the furnace without its touching the walls of the furnace door is critical to avoid contamination. The same microboat was used during the entire c o m of an analysis. Between samples, the pipet was rinsed with water and then with the next sample. Contamination or carry-over did not occur with this pipetting procedure. Recovery Studies. AA determination of Na and K in nanoliter samples was further evaluated by recovery studies and comparison of nanoliter determinations on IL951/655 versus microliter determinations using IL443 flame photometry on the same solution. Studies were carried out on nanoliter samples of tubular fluid collected by micropuncture from late proximal and early and late distal tubules. Aliquots of 0.1 nL of tubular fluid were delivered to the microboat and analyzed unaltered. Aliquots of an appropriate concentration, such that the total of standard plus sample would be within the range of the calibration curve, were likewise analyzed. Equal 0.1-nL aliquots of the tubular fluid sample-followed by the standard delivered directly on top of the sample on the microboat-were placed in the furnace and analyzed. Recovery was calculated as the totalelement determined in the sample plus the standard as a percent of the total element expected by the sum of sample plus standard when analyzed individually. Boric Acid Pretreatment. Reports by Sotera e t al. (7) and Krasowski and Copeland (8)suggested that boric acid pretreatment of the graphite platform could reduce interference by forming a boric carbide coating on the platform and thus prevent soaking of the sample into the graphite surface. Boric acid pretreatment of the microboat was evaluated for prevention of HCOBinterference with sodium. A 25-pL aliquot of 1% boric acid was delivered to the microboat and run through the same temperature program used for analyses with a 45-9 drying cycle. Boric acid treatment was carried out daily. Calibration. Figure 2 shows a typical standard curve for Na and K, reproduced with the Axiom EX-850 video printer from the video display on the IL951. The atomization absorbance vs time profile for a typical sample is also shown in Figure 2. Approximately 100 experiments involving analysis of tubular fluid samples were carried out in which Na and K were determined with this method. With appropriate choice of standards and
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988 K - 766.5nm 29 - 147 PS
Na 589nm 114 - 520 pg
Table 11. Comparison of Flame Emission of Microliter Samples Versus Atomic Absorption of 0.10 nL Samples
solution
A (n = 14) B (n = 15) C (n = 15) D (n = 10) E (n = 4)
A (n = 14) C (n = 15) D (n = 5) E (n = 4) Figure 2. Analysis of Na and K in 0.15-nL samples using IL951/655. Axiom EX-850 printer display shows representative potassium and sodium standard curves and atomization signal for a tubular fluid sample. In the upper part of the figure, peak area absorbance is plotted versus concentration (in mM).
sample volumes, repeat determinations of a given sample yield relative standard deviations (RSD’s)of less than 5%. A standard curve was usually generated for each set of five unknown samples. An automatic zero adjustment and a one-point autocalibration using a standard that was midpoint in the calibration line was carried out after each set of samples. A new standard curve was generated if the midpoint autocalibration was different by 20%. Analysis of an artificial tubular fluid sample was used as a quality control standard. Samples dried within 2 s of contact with the microboat, and therefore a 10-s dry cycle was considered adequate. Potassium determinations in the furnace rarely presented a problem, since potassium concentration in distilled water usually never exceeded the lowest concentration of potassium analyzed. Signals were similar for a sample volume of 25 pL or 0.1 nL, containing equivalent picograms of K. Determination of Na was complicated by the difficulty in obtaining Na-free water or reagents and by contamination problems. Although our water resistivity was 18.3 MQ cm-’, background was variable and often yielded higher absorbances than the nanoliter samples we analyzed. Knott (9)has shown that ultrapure water with a resistivity of 18.3 MQ an-’contains undetectable Na in free solution. Sodium may be present as oxide particulates and colloids or may be organically bound. This may in part account for some of the high sodium backgrounds we encountered. The ratio of signal to background was sufficiently high with a 0.1-nL sample volume to achieve low backgrounds and a large separation between the background and the lowest standard. However, 25 pL of H20may have more Na than 0.1 nL of the lowest Na standard, and the many possibilities of environmental contamination cannot be precluded. Subramanian and Chakrabarti (10) have reviewed the possible sources of contamination in trace element analysis, and thus exposure of the graphite platform alone to the laboratory environment is not without hazard for elements such as sodium and potassium. In our laboratory the AA was located in an area enclosed with glass walls in an attempt to reduce contamination. During analysis only the analyst was permitted access. The removal and replacement of the microboat for analysis was carried out in a slow methodical manner. Shaking the microboat, air currents or any dust can cause contamination.
RESULTS AND DISCUSSION Validation. Table I1 summarizes the comparison of flame emission analyses of microliter aliquots versus graphite furnace analyses of 0.1-nL aliquots for Na and K from the same 100-mL preparation or solution. For each experiment, Na and K were determined two or three times for each solution or sample, and the mean of these determinations was used as the value for that experiment. The values in Table I1 represent the mean concentration from n experiments. The relative error is the percentage difference between the assumed true value (FE) and the determination by AA. The value
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flame emission IL343 mean f SEM
atomic absorption mean f SEM
% re1 error
Sodium, mM 95.6f 0.30 94.8 f 0.55 94.9f 0.30 94.7 f 0.54 32.8 f 0.19 33.4 f 0.7 141.8 f 1.38 140.4 f 0.6 119.8f 0.3 118.6 f 3.4
-0.8 -0.2 +1.8 +1.0 -1.0
Potassium, mM 14.7 f 0.04 15.3 f 0.10 14.8 f 0.06 14.8 f 0.09 5.0 f 0.20 5.0 0.03 2.0 f 0.01 2.2 f 0.1
+4.0 0 0 +10.0
*
Table 111. Recovery of Sodium and Potassium from Tubular Fluid Samples
sample
recovery recovery potassium after std after std concn, addition, % sodium addition, % mM K concn,mM Na
proximal 4.2 f 0.19 99 f 1 tubule (n = 10) distal tubule 4.7 f 0.11 102 f 1 (n = 5) late distal 8.6 f 0.05 101 f 2 (n = 4) distal 4.5 f 0.11 97 f 3 distal
(n = 5) 1.6 f 0.09 100 f 4 (n = 6)
130 f 0.4 88.4 f 1.40 50.4 f 1.60 85.1 f 1.18 57.8 f 0.18
105 f 2 (n =lo) 104 f 1 (n = 5) 97 f 3 (n = 4) 108 f 3 (n = 5) 100 f 4 (n = 4)
determined by FE was considered the true value, since no quality control standards are available for trace volume (nanoliter) analyses for AA. In addition, for most physiological studies the ultramicro samples are compared with microliter plasma or urine samples determined by flame emission. I t should be emphasized that the variation in determinations represents the variation in daily analyses, taking into account variation in microsample handling as well as instrumentation. There is good agreement between flame emission of macrosamples and nanoliter samples that have undergone a number of manipulations. In addition, the assay value for Na or K in a 0.1-nL sample solution containing 95 mM NaCl and 15 mM KCl was not altered by increasing the concentration of urea up to lo00 mM nor of mannitol up to 250 mM (To convert millimoles of Na or K to picograms delivered to the microboat as a 0.1-nL aliquot, use: Na mmol/L X 2.3 = pg; K mmol/L X 3.9 = pg). The lissamine green dye used to identify different parts of the renal nephron contains sodium sulfate in a variable concentration (11). The sodium concentration of most of the solutions was increased by 5-8 mM due to presence of the dye. This explains the discrepancy between the values in Table I1 and those calculated from the composition of solutions. Although the comparison of flame emission of macrosamples versus atomic absorption of 0.1 nL clearly demonstrated the validity of the nanoliter assay for sodium and potassium, the possibility of interferences in tubular fluid samples could not be excluded. Recovery studies were carried out in representative tubular fluid samples into which an equal aliquot of 5 mM K and 30 mM Na was added to the microboat. Table I11 summarizes the results from five typical samples. Tubular fluid contains a number of unknown substances and often protein, which alters the viscosity of the samples. In addition,
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988 NaHCO,
NaCl
No,HPO,
Table IV. Na Absorbances for 200 pg of Na Compounds (589.6 nm)
salt
peak area"
NaCl Na,CO,
No, SO,
1.10 f 2.2% n=6 NaHCO, 1.19 f 0.20% n=2 Na2S04 1.02 f 1.5% n=4 Na2HP04 1.10 f 3.4% n=5 Na2C03 1.29 f 1.2% n=4 "Table shows peak area
peak height 0.66 f 0.7% n=6 0.75 f 3.1% n=2 1.24 f 2.8% n=4 124 f 0.2% n=4 0.78 f 1.0% n=4 f
difference in Na compared with NaCl std curve peak peak area height
+11.6%
+22.5%
-9.4%
+140.9%
-0.5%
+140.9%
+23.4%
+28.9%
RSD as a percentage of the mean.
Table V. K Absorbances for 90 pg of K in K Compounds Flgure 3. Sodium atomization signals for 200 pg of Na. Sodium
(766.5 nm)
atomization profiles demonstrate variability with various chemical forms of Na. there may be suppression or enhancement of the signal that may result from endogenous substances; thus recovery studies must be carried out. Anion Effects. All calibration curves for Na and K were generated by utilizing the chloride salts of these cations as standards. However, bicarbonate and other anions such as PO4 and SO4are present in tubular fluid, albeit at much lower concentrations. The Na concentrations of non-chloride Na salts were determined by using a NaCl standard curve to evaluate whether complete substitution of chloride by other anions would alter the determined value for Na. The atomization signals of approximately 200 pg of sodium are shown in Figure 3. As demonstrated, different sodium compounds had distinctly different atomization signals with the instrument parameters selected. However, in addition to these compounds, which are the most common anions found in tubular fluid, sodium acetate, sodium fluoride, trisodium citrate, disodium citrate, and sodium hydroxide were also analyzed. The results were not different from those for sodium chloride. Although the atomization signals were distinctly different, the peak areas were similar. Without the graphics display on the cathode ray tube (CRT), it would not have been as obvious that Na2HP04and Na2S04had not reached their peak absorbance and could not have been accurately assessed. However, peak area calculation would have given the same concentration as that of NaCI. To determine whether these differences were a result of a short pyrolysis cycle, analysis of these samples was repeated, using the methods described samples. The signal shape in the instruction manual for 2 5 - ~ L was similar to that described in the instruction manual for this instrument. The atomization signals were not altered, although absorbance was less; this may have been a result of loss of analyte. Complete substitution of chloride by other anions may affect the amount of sodium estimated from a sodium chloride standard curve. The peak area and peak height absorbances obtained for several compounds were compared to a sodium chloride standard curve. The error for both peak area and peak height was calculated as amount of sodium estimated from the NaCl curve divided by the known amount present expressed as a percentage difference, as shown in Table IV. Both peak area and peak height show differences between absorbances of NaCl and the other sodium salts listed.
salta
peak area
peak height
KC1 KHC03 KZSO, KzC03 KzHPO4
0.506 f 2.5% 0.509 f 0.0% 0.503 f 3.4% 0.524 f 1.8% 0.500 f 1.9%
0.404 f 2.0% 0.401 f 2.8% 0.472 f 2.0% 0.404 f 3.2% 0.451 f 5.0%
percentage difference from KCl std peak peak area height 0.6% -0.6% +3.6% -1.2%
-0.7% +16.8% 0.0%
+11.7%
a n = 4 for each salt.
Figure 4. Potassium atomization signals for 90 pg of K. Potassium atomization profiles demonstrate variability with chemical forms of potassium.
However, in tubular fluid samples over 75% of the sodium exists as sodium chloride, and complete substitution of chloride by other anions in the biological setting is unlikely. It would appear that peak area is the parameter least affected by the accompanying anion whereas there are dramatic differences in peak height shown for all compounds studied, when compared to that of NaCl. Atomization signals for approximately 90 pg of potassium in different potassium salts are shown in Figure 4. Quantitative differences in peak area and peak height are summarized in Table V. Potassium compounds did not show the
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
1.5
t
iLI
/-'?-
1.9 1.8 1.7
2417
7
-
NoHCO
Microboot II
I
0 90
10
80
25 65
a5 45
% ' haHCOm ,M
0 NaClmM
Figure 5. Effect of bicarbonate on sodium absorbance. Increasing the concentration of HCO, can increase the sodium signal, despite a fixed total sodium concentration.
same difference in peak height when chloride was replaced by other anions. Since only one mode may be selected when one is analyzing simultaneously in two channels, peak area was used for all subsequent determinations of potassium. Bicarbonate Effect. Besides the effect of bicarbonate depicted in Figures 3 and 4 and Tables IV and V, we also encountered an intermittent problem in determining Na in samples containing 25 mM or more of NaHCOS. Artificial tubular fluid solution containing 25 mM NaHC03 sometimes gave values for Na that were 10-40% higher than expected on the basis of a NaC1-KC1 standard curve. In these instances a linear increase in peak area absorbance as a function of bicarbonate concentration was found although the total sodium concentration was fixed. A typical example of this phenomenon is shown in Figure 5. Use of microboat I resulted in absorbance values with a coefficient of variation of 3.2%, 2.3%, 1.9%,0.4%,and 1.3% for 9, 10, 8, 6, and 6 determinations of solutions containing 0, 10, 25, 45, and 90 mM bicarbonate. The overall correlation coefficient for 0-45 mM bicarbonate concentration (within physiological range) was 0.9982 with a slope of 0.004 79 and intercept of 1.275. The correlation coefficient for 0-90 mM bicarbonate was 0.9489 with a slope of 0.00298 and intercept of 1.3045. This could represent as much as a 41 % apparent increase in determined sodium Concentration. Replacing the microboat in some circumstances could completely eliminate this phenomenon of the sodium signal with sodium bicarbonate. The range of our sample bicarbonate concentrations was 5-45 mM bicarbonate, determined by nanoliter total carbon dioxide analysis. As shown in Figure 5 , use of a new microboat I1 reduced the previously observed bicarbonate enhancement to a level where no significant difference in sodium peak area absorbance is detected unless NaCl is completely replaced by NaHC03. I t appeared that some component of aging or a physical chemical alteration in the platform surface may have been causal in this interference problem. Reports by Sotera et al. (7) and Krasowski and Copeland (8) suggested a possible resolution by treating the graphite surface with a solution of boric acid. The most extreme range of difference in the sodium signal found between NaHC03 and NaCl was assessed before and after boric acid treatment of the graphite platform. Figure 6 shows the alteration in NaCl and NaHC03 standard curves before and after boric acid treatment of the platform. These experiments demonstrate that quality control standards of similar constituents to the unknown samples is imperative to indicate the potential of erroneous determinations. Another possible cause for the interferences encountered in these experiments relates to the methodology used. Unlike manual applications of trace quantities in microliter volumes or the aerosol deposition technique, we deposited 0.1-nL volumes of a highly concentrated solution of complex matrices to the platform. This high concentration placed in a small area of the platform may increase the potential for large crystal
30
I
I
I
60
90
120
I
m M No
Figure 8. Boric acid pretreatment of microboats. Variation in peak area absorbance curves for Na is related to (a)the presence of chloride or bicarbonate as sodium salt and (b) pretreatment of the microboat with boric acid. Note that pretreatmentabolishes the disparity observed with the two different sodium salts.
formation and some soaking of the sample into the graphite. This may in turn result in the inteferences encountered (7, 8). Sodium-Potassium Interactions. Since the ratio of sodium and potassium concentration in tubular fluid samples may range from greater than 4:l (Na:K) to less than 1:1, we carried out studies to examine the effect of varying KC1 concentrations on the determination of Na in solutions with fixed NaCl concentrations. When NaCl concentration was fixed at 100 mM (230 pg of Na) while the potassium chloride concentration was increased to 200 mM (780 pg), the Na concentration estimated from the pure NaCl standard curve decreased linearly to 92.5 mM at a K concentration of 200 mM. When the NaCl concentration was fixed a t 50 mM Na (115 pg), no significant difference in the estimated Na concentration was observed with potassium chloride concentration up to 100 mM, but a t 250 mM (975 pg) KC1, an 8.5% reduction in determined sodium concentration was observed. While KCl suppressed the signal for 100 mM NaC1, equal concentrations of NHICl did not cause suppression; less volatile compounds were not studied. It was not ow intention to delineate the matrix effects with regard to K or C1 per se but to assess the limits to which we could expect reasonable assurance that tubular fluid samples containing high KCl concentrations could be measured. It is important to note that this method has been validated and routinely used for simultaneous determination of Na and K within the concentration range of 60-100 mM Na and 5-25 mM K. However, it is not certain that renal tubule fluid samples having concentrations of Na between 100 and 300 mM and of K between 10 and 150 mM can likewise be analyzed (e.g. collecting duct samples). The limiting factor, in laboratories where samples are not diluted, is the operator's ability to reproducibly deliver volumes less than 0.1 nL to the microboat. In these situations it may be necessary to dilute samples prior to analyses. Whether samples can be analyzed for Na and K simultaneously also depends on the expected concentration range for each element. For example, a 0.1-nL aliquot containing 150 mM sodium delivered to the microboat could not be accurately analyzed, whereas the same volume of a solution containing 150 mM K could be. On the other hand a K concentration less than 5 mM requires a 0.2-nL aliquot to ensure precision of 5% or less. It should be obvious that large differences in the ranges of Na and K would require that the determinations of Na and K be carried out in a single-channel mode, not simultaneously.
CONCLUSION Using graphite furnace atomic absorption spectroscopy, we have shown that nanoliter samples of tubular fluid can be
Anal. Chem. 1988, 60, 2418-2421
2418
assayed for Na and K simultaneously in 0.1-0.2-nL aliquots, usually without dilution, within the normal concentration range of tubular fluid samples. Bicarbonate ion has been shown to spuriously enhance the Na signal; a method of avoiding this error is described. Registry No. Na, 7440-23-5; K, 7440-09-7; HC03, 71-52-3; &Boa, 10043-35-3;NaC1,7647-14-5;NaHC03,144-55-8;Na$04, 7757-82-6;Na2HP04,7558-79-4 Na2C03,497-19-8; KCl, 7447-40-7; KHCO3, 298-14-6;K2S04,7778-80-5;KzCO3,584-08-7;K2HP04, 7758-11-4.
(4) Levine, D. Z.; Byers, M. K.; McLeod, R. A.; Luisello, J. A. J. Clin. Invest. 1979, 63. 59-66. (5) Levine, D. 2 . ; Roinel, N.; De Rouffignac, C. Kidney Int. 1982, 22, 634-649. (6) Paschen, K.; Fuchs, C. Clin. Chim. Acta 1971, 35, 401-408. (7) Solera, J. J.; Crislino, L. C.; Conky, M. K.; Kahn, H. L. Anal. Chem. 1983 55, 204-208. (8) Krasowski, J. A.; Copeland, R. R. Anal. Chem. 1979, 51, 1843-1649. (9) Knott, A. R. At. Absorpt. News/. 1975, 14(5). (IO) Subramanian, K. S.;Chakrabarti, C. L. Frog. Anal. At. Spectrosc. 1978, 2, 287-308. (11) Parekh, N.; P o p , G.; Galaske, R.; Galaske, W.; Steinhausen, M. Pfluegers Arch. 1973, 343, 1-9.
LITERATURE C I T E D
RECEIVED for review August 12,1987. Accepted June 6,1988. A portion of this work was presented a t the 29th Annual Conference of the Spectroscopy Society of Canada (1982). This work was supported by theMedical Research Council, Grant MT3836.
Kuntrlger, A.; Antonetti, S.;Couette, c.; Coureau, c.; Amiei, c. Anal. Chem. 1974, 46, 449-454. Good, D. W.; Wright, F. S.A m . J. Physid. 1979, 236(2), F192-F205. Vurek, G. G.; Bowman, R. L. Science (Washington, D . C . ) 1985, 749, 448-450.
Fluorescence Quenching Measurements of Copper-Fulvic Acid Binding Stephen
E. Cabaniss' a n d M a r k S . S h u m a n *
Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599- 7400
Current methodologies for calibration and analysis of fluorescence quenching (Fa)data cannot be recommended for studies of Cu binding by dissolved organic matter. Parameter estknates based solely on FQ data and a sknple 1:l model are unreliable due to strong covarlance among the three fltting parameters and large errors in computed [Cuz+] If total copper >> [Cu2+]. Addltional errors a r k because the relationship between quenchlng (0) and bound Cu concentration [CuL] Is not always linear as commonly asswned. Catlbratlng the fluorescence measurements Independently wHh a technlque more dlrectly related to metal concentratlon (as., potentlometry)Is recommended. Atthough fluorescence quenchlng is fast enough for use In klnetic studies, its error properties compare unfavorably with those of the CU-PAR spectrophotometric method In most cases.
Weber and co-workers ( I , 2) introduced fluorescence quenching (FQ) as a method for observing free ligand in copper equilibria with DOM (dissolved organic matter) cu2+
+ L + CUL
where L is a fluorescing ligand such as fulvic acid (FA). The concentration of bound copper, [CuL], is assumed to be proportional to the fluorescence quenched, Q (see Glossary for definitions). The method was originally validated with model ligands (I, 2 ) and compared to ion-selective electrode (ISE) measurements (I). Binding parameters estimated from FQ titrations were compared with those estimated from other analytical techniques (2). Several researchers investigated the underlying assumptions, error propagation, and potential usefulness of the method (2-4). Some authors reported dif-
* A u t h o r t o w h o m correspondence should be addressed.
C u r r e n t address: C h e m i s t r y Department, U n i v e r s i t y of N o r t h Carolina, Chapel Hill, N C 27599-3290.
ficulties in obtaining reasonable parameter estimates (2,5, 6). The present study investigates the causes of these difficulties; it employs FQ and Cu-ISE measurements to evaluate the FQ titration methodology and the usefulness of FQ for kinetic studies of Cu2+binding to a standard fulvic acid (FA). Fluorescence Quenching Titration Methodology. Saar and Weber (I)added Cu2+to soil FA solution a t pH 3-6 and found that Q was proportional to [CuL] calculated from ISE measurements. Ryan and Weber (2) describe a FQ titration methodology with which to estimate average binding parameters LT and K,, (defined in the Glossary). They tested the method on the model ligand tyrosine and on a soil FA a t three pH values and concluded that it could reliably determine these parameters. This method explicitly assumes that (1)the fluorescent fraction of DOM is representative of all the sample DOM, (2) the average parameters Kcu and LT adequately represent the data, and (3) 1:l stoichiometry applies. It also implicitly assumes that Q is proportional to [CuL], that is
Q = AfCuL] (1) where A is a constant with a value dependent on instrument setup as well as sample characteristics. Ryan and Weber used a nonlinear regression algorithm with CUTas the independent variable and fluorescence intensity, I , as the dependent variable to estimate the parameters LT,KcU,and Imin.This approach estimates A , since (2) A = (I,,, - Imin)/& Fluorescence quenching has several attractive characteristics for environmental studies. I t is fast, sensitive to low concentrations of DOM, and requires no supporting electrolyte. FQ complements metal detection techniques since it observes free ligand directly and the same instrumentation may be used for light scattering measurements (2). Fish and Morel (3) and Cabaniss and Shuman ( 4 ) noted the high relative error in Q at low [CuL]and the corresponding low error, relative to metal detection techniques, a t high [CuL]. Cabaniss and Shuman
0003-2700/88/0360-2418$01.50/00 1988 American Chemical Society
