Anal. Chem. 1986, 58,3028-3031
3028
Ion Chromatographic Procedure for Bicarbonate Determination in Biological Fluids Jack R. Kreling and Jack DeZwaan* 7255-209-0, The Upjohn Company, Kalamazoo, Michigan 49001
An Ion chromatographlc procedure for the determlnatlon of bicarbonate ion in biological flukls has been developed. This procedure Is capable of determfnlng blcarbonate levels down to around 0.1 mM In blood serum, blood plasma, bile, pancreatic julce, and s a k e duodenal petidon sdutlons and requires no sample preparation or handUng before analysls except for dHutlon with water. Careful evaluation of this chromatographk procedure has shown It to be highly selective for bicarbonate and to be free of Interferences for all biological fluids investigated. Additional advantages Include precision, small sample size requirements, rellablllty, and ease of operation.
The bicarbonate ion is a physiologically important species that is found a t significant levels in many biological fluids. The roles of bicarbonate as a means of carbon dioxide transport and p H management in blood have been extensively documented ( I , 2). Because of its clinical importance several means'including manometric (3),colorimetric (4,and enzymatic (5) procedures have been developed for bicarbonate quantification in blood serum. Bicarbonate also plays other important roles such as protecting the mucosa of the gastrointestinal tract (6,7). Because of the potential role of bicarbonate ion in the prevention and healing of ulcers (8,9),understanding the amount, distribution, and source of this material in the digestive system is an important area of research. The bicarbonate present in the duodenum, for example, can be influenced by a variety of chemical and biological agents and is typically measured by use of a back titration method on a saline (0.9%) perfusion solution (10). Because bicarbonate in the duodenum can arise from several sources such as the duodenal wall, bile, and pancreatic juice, a reliable means of analysis capable of accurately determining bicarbonate in a few microliters of these fluids is needed to fully characterize the response observed for any particular stimulus. A nonsuppressed ion chromatographic (IC) procedure using an ion exclusion column ( I I ) and a conductivity detector was found to provide excellent bicarbonate determinations when applied to this entire range of biological fluids. The IC procedure was directly compared with the back titration procedure and found to be more generally applicable because the IC bicarbonate signal was free from interaction with the matrix and produced a more readily quantifiable response. In addition to high selectivity, other potential advantages of the IC procedure over current methods of analysis include sample size (1-3 &), ease of operation (no sample preparation except for dilution with water), and precision (2-3% relative standard deviation).
EXPERIMENTAL SECTION Chromatography was done with a Dionex Model 2010i ion chromatograph with a conductivity detector (Dionex Corp., Sunnyvale, CA). The chromatographic conditions used are given in Table I. No suppressor column or device for lowering conductivity was used in any of the work reported. An Upjohn
Table I. Chromatographic Conditions
parameter column eluent flow rate
pressure detection injection temperature
description Dionex HPICE-AS1 double-distilled water that was degassed and continuously purged with helium 1.5 mL/min 1330-1400 psi
electrical conductivity 50 fiL
ambient
autosampler was used for multiple sample injection, and the data generated were digitized (1point/s) and stored on a Harris H800 computer. Bicarbonate determinations by the back titration method were done by adding excess sulfuric acid (0.0125 N) to the sample and then degassing by heating to boiling. The samples were then titrated with potassium hydroxide (0.25 N) to an equivalence point using a Brinkman Model E436 potentiograph and a Corning 476050 combination electrode. Stock standard solutions were prepared by dissolving sodium bicarbonate (Mallinckrodt) in double-distilled water at the following approximate concentrations: 500, 1000, 1500, 2000, 2500, 3000, 4000, and 5000 bg of bicarbonate/mL of water. Final standards were made by making 1:50 dilutions of the stock solutions. The shelf life of these stock standard solutions has been several months.
RESULTS AND DISCUSSION The chromatogram of a standard bicarbonate solution obtained using this system is shown in Figure 1. This chromatogram may be compared with the other chromatograms in Figures 1 and 2, which are representative of the sample types investigated. No sample preparation of the biological samples was involved except for dilution with double-distilled water. The dilutions used were typically 1:50 or 1:lOO for pancreatic juice and bile and 1 : l O for duodenal perfusion solutions. By use of this chromatographic system, anions of all strong acids, (e.g., chloride, sulfate, nitrate) are unretained, elute a t the solvent front, and are well-resolved from the bicarbonate peak. The peak height respones obtained as a function of bicarbonate concentration for standard solutions are given in Figure 3. These data demonstrate that a transition from double-distilled water, which was degassed and purged with helium (lowest conductivity), to deionized tap water, which was used directly (highest conductivity), can have a significant effect on the observed response. An effect of the same type and magnitude can be induced by adding sub-part-per-million levels of NaCl or KI to the double-distilled degassed water and using this solution as the mobile phase. I t is evident from Figure 3 that the measured bicarbonate response is not necessarily a linear function of concentration and that the purity (conductivity) of the mobile phase can influence the observed response. These were not found to be significant problems, however, since calibration curves were used to determine concentration and a single reservoir of mobile phase was used throughout the course of an entire series of samples to assure a constant response. Constancy
0 1986 American Chemical Society 0003-2700/86/0358-3028$01.50/0
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
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Table 11. Peak Height Responses of Bicarbonate Standard Solutions as a Function of Time" bicarbonate std concn, ppm
10
1
RETENTION TIME (MINUTES)
Flgure 1. Ion chromatograms obtained for a 1 mM bicarbonate and for blood plasma from a rat (---) show the standard (-) bicarbonate response at 8.4 min. The base lines were artificially offset.
time, h
10
40
100
0 5 10 15 25 av re1 std dev %
39 535 38419 38 096 38 278 38 767 38 619 1.47
140 390 139 530 137 848 140 307 140518 139 719 0.80
286 873 285 228 287 100 289 567 288316 287 417 0.57
"Arbitrary units. A single reservoir of double-distilled water, degassed and continuously purged with helium, was used. Table 111. Comparison of Results Obtained for Bicarbonate Concentration by the Standard Additions and Calibration Curve Methods
bicarbonate concn, mM calibration
sample type
std additions
curve
duodenal perfusion solution
5.6 4.2 4.6 35 29 31 32 33 36 20 18
5.9 4.5 4.3 32 28 31 31 31 36 20 18
pancreatic juice (rat)
bile (rat) 10
1
RETENTION TIME (Minutes)
serum (rat) plasma (rat)
Figure 2. Ion chromatograms obtained for saline duodenal perfusion solution from a rat (-) and for bile from a dog (---). The base lines were artificially offset.
II -
E Ly r Y
d n / /
-40
BICARBONATE CONCENTRATION (MILLIMOLAR)
Figure 3. Calibration curves obtained using water of various conductivities as the mobile phase. Shown a r e doubledistilled and degassed (0),mixture of double-ditilled and deionized (O),and deionized tap water (0).
of response for this assay for a period of time exceeding 24 h is demonstrated in Table I1 using a single reservoir of double-distilled water, which was degassed and continuously purged with helium. Checking the instrumental response with a series of standards to assure proper calibration is recommended after changing or recharging the mobile-phase reservoirs during the course of a determination because of the sensitivity of this method to certain types of trace contaminants in the mobile phase. The biological fluids that were investigated are listed in Table I11 along with the bicarbonate levels determined by a
I
I
!
I
I
I
20 40 60 80 BICARBONATE CONCENTRATION (MILLIMOLAR) FROM ADDITIONS -20
0
I
100
Figure 4. Standard additions to a water blank (0)and to a sample This demonstrates the equivalence of of rat pancreatic juice (0). results obtained using a calibration curve (- - -) and the standard addition extrapolation (- - -).
calibration curve procedure and a standard additions procedure. These procedures are illustrated in Figure 4 where responses obtained from standard additions to a sample of and a water blank ( 0 )are plotted against pancreatic juice (0) the concentration of added bicarbonate standard solution before dilutions. The filled circles produce a calibration curve that allows the concentration of the pancreatic juice to be determined directly from the sample to which no bicarbonate was added (dotted lines). The shape of this curve can then be used to extrapolate the pancreatic juice responses to the intercept (dashed line). Excellent agreement between these two methods was obtained for all sample types as shown in Table 111. Although the additions method is more difficult and requires more sample, the results obtained are not influenced
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
Table IV. Comparison of Results Obtained by IC and Back Titration Methods for Bicarbonate Concentration Determination
sample type
sample no
40 mM standard 60 mM standard duodenal perfusion solution (rat)
bile (dog) pancreatic juice (dog) pancreatic juice" (rat) blood serum (rat) blood plasma (rat) a
1 1 1 1 1 2 3 4 1 2 3 1 2 1 2 3 1 1
bicarbonate concn,mM IC titration 39.8 39.7 60.1 60.0 23.3 18.3 17.4 15.8 53.1 54.4 49.2 138 132 65.6 52.0 50.2 20.3 17.8
38.8 39.0 61.7 61.0 25.9 20.0 19.4 15.9 51.0 50.2 45.2 115 106 63.0 48.0 50.1 19.8 14.9
Secretin stimulated.
Table V. Bicarbonate Concentration in Pancreatic Juice (Rat) Basal and Secretin Stimulated bicarbonate concn, mM before stimulation after stimulation
rat no.
22.5 24.6 20.2 22.5 22.6 22.3
1 2 3 4 5 6
65.6 48.5 52.0 52.0 58.4 50.2
Table VI. Precision Obtained upon Repeated Analysis of a Sample of Rat Bile and Duodenal Perfusion Solution trial
bicarbonate concn, mM rat bile duodenal s o h
1 2 3 4 5 6 7 8 9 10
27.9 29.2 29.1 29.5 28.3 28.9 29.2 29.2 29.1 29.7
5.8 5.6 6.0 5.9 5.7 5.9 5.8 6.0 6.0
av
29.0 1.9
5.84 2.4
RSD"
5.1
'Relative standard deviation (70).
~
KOH
ADDED-
Figure 5. Back titration curves obtained from dog bile (- - -) and dog pancreatic juice (-).
by any potential effects of the biological matrix on the bicarbonate response (peak height). Because excellent agreement was obtained for all sample types between the two methods, it follows that there is no significant effect of the biological fluid on the observed bicarbonate response. Since no matrix effects were observed, all other bicarbonate determinations reported were obtained by use of the simpler calibration curve procedure. The IC method and back titration method for bicarbonate determination were compared directly for a series of samples. The results obtained are given in Table IV. The agreement obtained was good, for standard solutions and most samples, although significant differences were observed for a couple of samples. The reasons for these differences have not been completely established but they arise a t least in part from uncertainty in locating the equivalence point on the titration curve. This is illustrated in Figure 5 where a titration curve with a clearly defined equivalence point (dog bile) is compared with a titration curve obtained from a sample of pancreatic juice (dog). Because of the gradual pH change around the equivalence point and the distortion of the curve itself, significant and systematic error in the bicarbonate level determined could arise using the back titration method. An even more gradual pH change around the equivalence point was found for the plasma and serum samples studied. Because
the bicarbonate response obtained by IC was clearly resolved from other peaks and because no influence of any particular matrix on the measured response could be found, IC appears to offer a significant advantage over back titration for these types of samples. Table IV shows the bicarbonate concentrations of pancreatic juice obtained from anesthetized rats both before and after administration of secretin (25 pg/ kg, subcutaneously). Secretin is known to stimulate both the volume of pancreatic juice produced and the bicarbonate concentration in the juice. The expected increase in concentration is clearly demonstrated in Table V. Multiple assays of these samples indicated that the samples, after dilution, were stable with respect to bicarbonate determination over the course of an entire day if kept in covered containers. These repetitive analyses indicated that a relative standard deviation of 2-3% was obtained. This precision is consistent with that obtained for standard solutions as well as the bile solutions and saline (0.9%) perfusion solutions reported in Table VI. Precision for samples with a bicarbonate concentration of less than 1 mM decreased somewhat to a relative deviation of around 5%. No bicarbonate levels of less than 0.2 mM were encountered.
ACKNOWLEDGMENT The support of Andre Robert and his associates from the Gastrointestinal Diseases Unit of The Upjohn Company in providing biological samples and assistance with this work is gratefully acknowledged. Registry No. HC03-, 71-52-3. LITERATURE CITED (1) Shapiro, B. A.; Harrison, R. A.; Walton, J. R. Clinical Application of Blood Gases; Year Book Med.: Chicago, IL, 1977; pp 1-39. (2) Winters, R. W.; Engel, K.; Dell, R. B. Acid-Base Pbyslology in Medicine; The London Company: Cleveland, OH, 1967; pp 44-48, and 279-286. (3) Van Slyke, D. D.; Neili, J. M. J . Biol. Cbern. 1924, 61, 523-528. (4) Sterling, R. E.; Flores, 0. R . Clin. Cbem. (Winston-Salem, N.C.) 1972, 18, 544-547.
Anal. Chem. 1886, 58,3031-3035
(5) Forrester, R. L.; Wataji, L. J.; Siiverman, D. A.; Pierre, K. J. Clin.
Chem. (Winston-Salem, N.C.) 1978, 22, 243-245. (6) Forsseli, H.; Olbe. L. S a n d . J . Gastroenterol. 1985, 20, 767-774. (7) Isenberg, J. I.; Flemstrom, 0.;Johansson, C. Mechanisms of Mucosal Protection in the Upper Gastrohrtestinal Tract: Allen, A., Ed.; Raven: New York, 1984; pp 175-180. (8) Kiviiaakso, E.; Flemstrum, G. Mechanism of MucosalProtection in the Upper Gastrointestinal Tract; Ailen, A,, Ed.; Raven: New York, 1984; pp 227-232,
3031
(9) Tabata, K.; Jacobson, E. D.; Chen, M.; Murphy, R. F.: Joffe, S. N. Gastroenterology 1984, 87, 396-401. (10) Isenberg, J. I.; Wallis, E.; Johansson, C.; Smedfors, E.; Mutt, V.; Tatemoto, K.; Emas, S. Regul. fept. 1984 3, 315-320. (11) Smith, F. C., Jr.; Chang, R. C. The Practice of Ion Chromatography; Wiiey: New York, 1983; p 132.
RECEIVED for review May 15, 1986. Accepted August 11, 1986.
Vinyl Polymer Agglomerate Based Transition Metal Cation Chelating Ion-Exchange Resin Containing the 8-H yd roxyquino1ine FunctionaI Group William M. Landing,*’Conny Haraldsson, and Nicklas Paxgus Department of Analytical a n d Marine Chemistry, Chalmers University of Technology and University of Goteborg, 8-412 96 Goteborg, Sweden
A simple synthetic route has been developed for the ImmobHlzation of 8-hydroxyqulnollne onto Fractogel TSK, a highly porous, mechanlcally and chemically stable, hydrophlllc organic resin gel. The product exhiblts an exchange capacity comparable to the hlghest values reported for slllca-lmmobillzed l-hydroxyqulnollne but Is more stable at hlgh pH. The resin’s selectlvlty and efflciency of coilectlon of catlonlc metal specles from freshwater and seawater were Investlgated. The resin was used In a column sequence to obtain concentration and spedatlon data for AI, Mn, Fe, Co, Cu, Zn, and Cd In an organic-rlch freshwater sample.
The chelating qualities of 8-hydroxyquinoline (also known as 8-quinolinol or oxine, and hereafter referred to as 8HQ) and its preference for transition- and heavy-metal cations relative to alkali and alkaline-earth cations are well-known. These properties have led to a significant research effort in order to suitably immobilize this chelating agent onto various solid supports to utilize it in ion-exchange or chromatographic applications. A number of methods for the immobilization of 8HQ onto silica substrates (silica gel or glass beads) have been proposed (1-3), modified, and optimized (4-7), and the characteristics of the various products have been investigated (7-13). Silica supports offer the advantages of good mechanical strength, resistance to swelling, and rapid overall exchange kinetics in column applications (11). However, they are unstable at high pH, leading to cleavage of the immobilized 8HQ and potential trace-metal contamination from the newly exposed silica surface (2, 3, 11). The preparation of condensation resins of the resorcinolformaldehyde8HQ type has also been reviewed (14),and their properties have been further investigated (14-1 7). These resins offer higher exchange capacities, but low stability in acid solution and slower overall kinetic exchange rates. The immobilization of 8HQ onto polystyrene-divinylbenzene has also been investigated (14, 15, 17-19). These resins are apparently quite stable with respect to extremes of p H and can *Present address: Department of Oceanography, Florida State University, Tallahassee, FL 32306.
be produced with high total exchange capacity. Their overall kinetic exchange rates are also reported to be slow (15); however they have been successfully used in column applications at flow rates up to 16 mL/min (19). The polymer substrate used in this investigation, Fractogel-TSK, consists of intertwined vinyl polymer agglomerates, which offer mechanical and chemical stability, high porosity, and high hydrophilicity due to the presence of ether linkages and hydroxyl groups. The hydroxyl groups (as secondary alcohols) also enable relatively simple chemical modification, and this feature is used in the immobilization of 8HQ via phenyl-azo linkages. The preparation time is relatively short (>20 h) and yields a chelating resin with exchange capacities comparable to the highest values reported for silica-immobilized 8HQ. The polymer itself exhibits no cation exchange capacity and does not concentrate dissolved organic species such as humic or fulvic acids. The following properties of the Fractogel-immobilized 8HQ product were investigated: stability with respect to extremes of pH; exchange capacity with respect to pH and flow rate; and trace metal cation collection efficiency from a seawater reference material. The modified resin was further utilized to investigate speciation and quantify concentrations of trace metals in prepared seawater solutions and in an organic-rich freshwater sample.
EXPERIMENTAL SECTION Reagents. All reagents were analytical grade and were used as received unless otherwise specified. Intermediate-purity water was prepared by distillation of deionized water by use of a glass still equipped with a quartz immersion heater. High-purity water was prepared in a “clean”laboratory (positive-pressureClass-100 filtered air supply) using a Milli-Q (Millipore)system. High-purity acids were prepared in the clean lab by subboiling point quartz distillation of analytical grade acetic acid, HCl, and HN03. Fractogel TSK HW-75(F) (32-63 pm bead diameter, -7.5 nm pore diameter) was obtained from E. Merck and prepared in the following manner. A 50-mL volume of the resin slurry was washed three times with 100 mL of water to rinse away the NaN3 present as a preservative. The supernatant from each rinse was discarded after allowing the resin beads to settle for 30 min. The resin was then vacuum filtered onto a Whatman GF/F glass microfiber filter and rinsed with the following sequence of reagents: two 50-mL portions of 1.0 M NaOH; three 50-mL portions of H20;two 50-mL
0003-2700/86/0358-3031$01.50/00 1986 American Chemical Society
