Anal. Chem. 1980, 52,864-869
upon the results of this study (33).
LITERATURE CITED (1) Lane, R. F.; Hubbard, A. T. J. Pbys. Cbem. 1973, 77, 1401-1410. (2) Brown, A. P.; Koval, C.; Anson, F. C. J. Nectroanal. Cbem. 1978, 72, 379-387. (3) Moses, P. R.; Wier, L.; Murray, R. W. Anal. Cbem. 1975, 47, 1882-1886. (4) Moses, P. R.; Murray, R. W. J. Am. Chem. SGC. 1978, 98, 7435-7436. (5) Moses, P. R.; Murray, R. W. J. Nectroanal. Cbem. 1977, 77, 393-399. (6) Lenhard, J. R.; Murray, R. W. J. €kcfroanal. Cbem. 1977, 78, 195-201. (7) Elliott, C. M.: Murray, R. W. Anal. Chem. 1978, 48, 1247-1254. (8) Evans, J. F.; Kuwana, T. Anal. Cbem. 1977, 49, 1632-1635. (9) Watkins, B. F.; Behling, J. R.; Kariv, E.; Miller, L. L. J. Am. Cbem. Soc. 1975, 97, 3549-3550. (10) Mazur, S.;Matusinovic, T.; Cammann, K. J. Am. Chem. SOC.1977, 99, 3888-3890. (11) Oyama, N.; Brown, A. P.; Anson, F. C. J. Nectroanal. Cbem. 1978, 87, 435-441. (12) Nowak, R.; Schultz, F. A,; Umana, M.; Abruna, H.; Murray, R. W. J. Nectroanal. Cbem. 1978, 94, 219-225. (13) Oyama, N.; Yap. K. B.; Anson, F. C. J. Nectroanal. Cbem. 1979, 100, 233-245. (14) Lin, A. W. C.; Yeh, P.; Yacynch, A. M.; Kuwana, T. J. Nectroanal. Cbem. 1977, 84, 411-419. (15) Miller. L. L.; Van de Mark, M. R. J. Am. Cbem. Soc. 1978, 700, 639-640. (16) Brown, A. P.; Anson, F. C. J. Nectroanal. Chem. 1977, 83, 203-206. (17) Koval, C. A.; Anson, F. C. Anal. Cbem. 1978, 50, 223-229. (18) Oyama, N.; Anson, F. C. J. Am. Cbem. SOC.1979, 107, 1634-1635.
(19) Oyama, N.; Anson, F. C. J. Am. Chem. Soc. 1979, 101, 739-741. (20) Wier, L. W.; Murray, R. W. J. Necfrochem. SOC.1979, 126, 617-623. (21) Lennox, J. C.; Murray, R. W. J. Am. Chem. SOC. 1978, 100, 37 10-37 14. (22) Rocklin, R. D.; Murray, R. W. J . Electroanal. Cbem. 1979, 100, 271-282. (23) Brabec, V.; Kim, M. H.; Christian, S. D.; Dryhurst, G. J. Nectroanal. Cbem. 1979, 100, 111-133. (24) Angerstein-Kozlowska, H.; Conway, B. E.; Sharp, W. B. A. J. Nectroanal. Cbem. 1973, 43, 9-35. (25) Christie, J. H.; Osteryoung, R. A,; Anson, F. C. J. Electroanal. Cbem. 1987, 13, 236-244. (26) Pearson, R. G. J. Cbem. Educ. 1988, 45, 581-587, 643-648. (27) Martin, R. B.; Mariam. Y. H. In "Metal Ions in Biological Systems", Vol. 8, Sigel, H. Ed.; Marcel Dekker: New York, 1979; Chapter 2. (28) Rabinowitz, I. N.; Davis, F. F.; Herbert, R. H. J. Am. Cbem. Soc. 1988, 88, 4346-4354. (29) Laviron, E. Bull. SOC.Cblm. Fr. 1987, 3717-3721. (30) Laviron. E. J. Electroanal. Cbem. 1979, 100, 263-270. (31) Laviron, E. J. Electroanal. Cbem. 1974, 52, 355-393. (32) Kuo, K.; Moses, P. R.; Lenhard, J. R.; Green, D. C.; Murray, R. W. Anal. Cbem. 1979, 51, 745-748. (33) Cox, J. A.; Majda, M.; unpublished results, 1979.
RECEIVED for review November 19, 1979. Accepted January 29, 1980. Partial support for this work was received from the Eastern European Universities Exchange Program grant from the U.S. State Department to SIU-C.
Fiber Optic pH Probe for Physiological Use John I. Peterson,* Seth
R. Goldstein, and
Raphael V. Fitzgerald
Biomedical Engineering and Instrumentation Branch, Division of Research Services, Building 13, Room 3 W- 13, National Institutes of Health, Bethesda, Maryland 20205
Delwin K. Buckhold Section on Laboratory Medicine and Surgery, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
20205
A flber optic probe has been developed for pH monitoring, based on the use of a dye indicator. Microspheres of polyacrylamlde containing bound phenol red and smaller polystyrene microspheres for light scattering are packed In an envelope of cellulosic dialysis tublng at the end of a pair of plastic optical flbers. The probe measures pH over the physiological pH range of 7.0 to 7.4 to the nearest 0.01 pH unit. It is of flexible construction and is about 0.4 mm in diameter.
A non-electrical p H probe, based on fiber optics and a dye sensor, offers some advantages in physiological applications over the conventional micro-pH electrodes which are available. An important safety feature for human use is that no possible electrical connection to the body is involved. The use of a plastic optical fiber allows a high degree of mechanical flexibility combined with very small size and low-cost, disposable construction. A probe has been designed which is suitable for tissue and blood p H measurements over the physiological range of pH 7.0 to 7.4 with an accuracy of 0.01 p H unit. The probe can be inserted into tissue or a blood vessel through a 22-gauge (0.41-mm i.d.) hypodermic needle. It can be used for monitoring pH in studies of respiration and tissue oxygenation, and was originally developed for obtaining the pH value necessary
for fixing the position of the blood oxygen saturation curve, which shifts with p H (Bohr effect). The soft construction makes the probe potentially suitable for muscle implantation in exercise experiments. It can also be inserted into a blood vessel for monitoring p H during operative procedures. Principle of pH Measurement. The probe is based on the use of the indicator dye phenol red (phenolsulfonphthalein). In the p H range of interest, this dye behaves as a weak acid of pK 7.9 and exists in two tautomeric forms, each having a different light absorption spectrum (1). As the p H of the solution varies, the relative size of each tautomer's optical absorption peak varies in proportion to the changing relative concentrations of the acid and base forms of the dye. Thus the optical absorption of the dye solution at one of these peak wavelengths can be used for measuring pH. pH can be expressed as a function of the pK of the indicator, the total dye concentration (T),and the concentration of the base form of the indicator (A-): PH = p K
-
log[ -
11
The base form is chosen because its optical density is greater than that of the acid form, thus providing a better optical sensitivity to p H changes. For the purpose of making a fiber optic probe measuring instrument, it was desirable to extend this familiar theory to
This article not subject to U.S. Copyright. Published 1980 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
865
the relation between the intensity of transmitted light a t the peak absorption wavelength of the base form of the dye and p H , in order to develop a relation which allows a simple instrument design for the p H measurement. Substitution into Equation 1 of the Beer-Lambert relation for optical density (Equation 2) log
(+)
= (A-)Lt
where I and Io are the transmitted intensities at the absorption wavelength, and Io represents the transmittance in the absence of any base form of the dye, L the length of the effective light path through the dye, and t , the absorption coefficient of the base tautomer, provides Equation 3
r
1
1
Here p H is related to I , the transmitted green light intensity a t 558 nm, the base peak, and constants of the probe. T h e constants (I"),e , a n d L can be combined into a constant C, which is the optical density which results from the dye being totally in the base form (see Equation 2). We define (Equation 4)
R'=
[ i]
(4)
green
for measurement reasons which will be evident later, and for convenience (Equation 5 ) :
A = pH
-
pK
(5)
As a result, we have Equation 6:
One further consideration must be entered. In the usual double-beam spectrophotometric measurements, the reference intensity Io is measured at the same wavelength as the sample transmitted intensity I. In the p H probe, a reference intensity measurement is needed to compensate for optical and instrument variations (hence the ratio R ' i n Equation 4), but it is not practical to measure the green light that would be transmitted with no dye in the base form. The only necessary condition for measuring Io is that it have no p H dependence, so it can be measured at any light wavelength where this is the case. This can be either the isosbestic point between t h e acid and base absorption peaks, a t 478 nm, or, as is preferable for instrumentation reasons, a t any wavelength longer than 600 nm. The instrument was designed to measure I,,& at 600 nm and Igreen at 558 nm, and combine them in the ratio R (Equation 7)
R = Igreen/IO,d
(7)
Iomn and IOrd are both independent of p H and in fixed proportion k to each other for a given probe, so (Equation 8): and, from Equations 4,7 , and 8,
R = kR' So, finally, Equation 10
R = k x 10[-c/(lO-A+
111
(10)
relates the green t o red intensity ratio R measured by the
Figure 2.
I-",
Probe construction
instrument to p H and constants of the probe C, k , and pK. Figure 1 shows the relation between R and A for t h e situation where a dye behaving as a weak acid, such as phenol red, dissociates into acid and base forms; the left curve is for measurement of the base form of the dye, and the right curve is for observation of the optical absorption of the acid form of the dye. T h e latter curve is given by substitution of +A for -A in Equation 10. I t can be seen t h a t a n S curve with a nearly linear center portion forms, suitable for linear signal processing. Because the ends of the curve approach R = 12 and R = k X k and C can be determined by measuring R a t extremes of A. T h e straight portions of t h e curves do not coincide with S = 0, as might be expected, b u t depend on the value of C, determined by t h e characteristics of the probe. T h e inflection points lie a t Sinfl.
= flog
[
-1
(C In 10) + [(C In 10l2 + 411/' 2
(11)
and spread farther from S = 0 as C increases. T h e value of C can be varied, along with the pK of the dye chosen, to place the straight portion of the R vs. A curve close to t h e range of p H measurement of interest. Increasing C will increase the slope and thereby the measurement sensitivity, b u t a t t h e expense of increasing t h e curvature and narrowing the p H range of reasonably linear response of R , and also decreasing probe brightness (smaller I,,,), which decreases t h e signalto-noise ratio. A value of C of approximately 1 is a good balance between sensitivity, brightness, and width of useable linear range for measurement to the nearest 0.01 p H unit, resulting in the inflection point occurring a t a p H about 0.5 from the pK. T h e curve shown in Figure 1 is for 12 = 0.7, C = 1, with inflection points at A = f0.43.
EXPERIMENTAL Construction of the pH Probe. Figure 2 shows the design of the probe. It is made by inserting a pair of 0.15-mm diameter plastic optical fibers (Welch-Allyn Company, Skaneateles Falls, N.Y. 13153) through 3-5 cm of Cuprophan hollow fiber (#B4AH, Enka Glanzstoff AG, P.O. Box 15291, Charlotte, N.C. 28210). This cellulosic dialysis tubing has a 0.30-mm i.d. and a 0.019-mm wall thickness. It must be rinsed thoroughly by suction with dry ethanol and air, t o remove the isooctyl alcohol filling. After inserting the optical fibers through the desired length of dialysis tubing (most of this length serves as a protective sleeve above the probe and is optional), the fibers and sleeve are cut square with a sharp razor blade. The fibers are then pulled back into
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
the tubing about 4 mm, with the optical fiber ends beside each other. The 4-mm empty space at the end of the tubing is then packed with indicator, and a small amount of optical adhesive ($60, Norland Products, Inc., P.O. Box 145, North Rrunswick, N.J. 08902) is applied to each end of the cellulosic tubing to provide a seal. The optical adhesive is set by exposure for 1 min to the surface of a long-wave hand ultraviolet lamp with a 4-W mercury vapor bulb (Mineralight UVSL 25, Cltravioiet Products, Inc., San Gabriel, Calif. 91778). I t is generally desirable to enclose the optical fibers in a protective sleeve for most of their length, from the probe to the connection with the measuring instrument. Black Teflon tubing is suitable for this. The twin optical fibers can be enclosed in 2&gauge lightweight spaghetti. and the single opticnl fibers, where they connect individually to the instrument, can be fitted with 30-gauge thin-wall spaghetti (Chemplast, Inc., 150 Dey Road, Wayne, N.J. 07470). A short length of 1/16-iiich(1.6-mm) i.d. poly(viny1 chloride) heat-shrinkable tubing can be used to cover the joint where the paired fibers branch, and this holds the protective tubing in place on the optical fibers. At the ends where connection to the instrument is made, the optical fiber is cut square with a razor blade so that about 2 inm protrudes from the protective Teflon tubing. The end of the optical fiber is then brought close to a hot soldering iron to form the fiber end into a flare, about 0.4-mm diameter, which increases the optical aperture substantially and prevents the fiber from pulling back inside the tubing. This completes the assembly. Probe Packing Material. The heart of the probe is the pH sensitive indicating packing which fills the hydrogen-ion permeable cellulosic tubing. The tubing serves the purpose of providing mechanical containment for the indicator and allows diffusion of hydrogen ions and small molecules, but blocks large molecules and cells. The packing consists of dyed polyacry1amid.e microspheres of diameter mostly in the 5- to 10-pm range. The dye must be fixed to some support to prevent it from diffusing out of the probe and to provide a simple means of packing the probe with dye. The polyacrylamide microspheres provide an open hydrophilic gel structure for ionic diffusion in the presence of the dye, which is covalently bound to it. Phenol red was chosen as the indicator dye because its pK is appropriate and toxicological hazards would not be expected. The polyacrylamide microspheres also contain smaller polystyrene microspheres of approximately I-fim diameter. These provide a very effective light scattering, so that the light which is emitted from one optical fiber passes into the packing, is absorbed by the dye to a degree dependent upon pH, and by multiple scattering is returned to the other optical fiber for transmission to the measuring instrunlent. 'This results in a miniature spectrophotometric cell, without the necessity of having the fibers in a difficult in-line-opposed construction. The probe packing contains approximately 20% polystyrene microspheres and 1%phenol red by weight, representing 1 dye molecule for 425 acrylic units on the average. The amounts of dye and reflecting microspheres used in the packing were selected by optimization experiments on probe performance. The following emulsion polymerization method is used to make the probe packing. Prepare the following solutions in two 13 mm X 100 mm test tubes. Solution 1: add 0.5 ml, of a stock solution cont,aining 6 M acrylamide (Eastman X5521), 0.06 M N,h'-methylencbisacrylamide (Eastman 8383), and 5 mg/mL phenol red (Eastman 541 or La Motte 2411). To this, add 0.5 mL polystyrene microspheres, 0.945-rm diameter, 10% m/v suspension (Dow Chemical Co.). Dissolve approximately 25 mg ammonium peroydisulfate (Eastman 11151) in this mixture. Solution 2 consists of 2 drops of N,N,N 'a'-tetramethylethylenediamine (Eastman 8176) aclded to 5 mL of toluene containing 0.25 g of dissolved emulsifier; the emulsifier is a mixture of 85 parts Span 80 and 15 parts Tween 80 by weight (IC1 United States, Inc.). Deoxygenate solutions 1 and 2, in test tubes, by bubbling nitrogen through each for 5 min. Add solution 2 to solution I, cover with nitrogen, stopper, and shake vigorously until polymerization occurs, noted by the development of heat and color change, which should be in 1 to 2 min. Let stand 15 min and wash the resulting precipitate, using a centrifuge for separation: three times with 1:l ethanol:water, with sodium hydroxide added RS
needed to make the dye basic (purple); then once with the same wash, but made acidic (orange) with acetic acid added; then wash twice with absolute ethanol. Filter by suction, using absolute ethanol as a wash, and dry by air flow through the filter. Dye Copolymerization. Initially an attempt was made to bind the dye to polyacrylamide or other supports, using the usual derivative technology in which a dye molecule is bound to a matrix by an intermediate chemical link. It was discovered that dyes of some pH indicator types (phenol red, brilliant yellow CI24890 (Eastman 837), and rosolic acid (2143800 (Matheson Coleman and Bell RX180/NB401) were tried), when present in the solution of acrylamide, copolymerize with it and become fixed to the acrylamide polymer. This provides a useful means of converting water soluble indicator dyes into a water insoluble form, and may be useful for purposes other than the pH probe. The chemical and patent literature does not disclose this possibility of direct copolynierization of dyes without derivative links attached. The dye must be covalently attached to the vinyl polymerized chain for the following reasons: (1) Ring attachment must be the site of binding, becauve any other kind of attachment would destroy the indicator function. (2) A dye similar to phenol red (bromthymol blue) which has its indicator ring positions occupied will not bind to the polyacrylamide. Apparently the third sulfonated ring is not the binding site. (3) Extensive aqueous washing, including acid and base which would cause hydrolysis, does not remove the dye. (4) The pK of the dye is shifted noticeably, from the usual value of 7.9 for soluble phenol red, to 7.57 (see below). This is significant because, among the triphenylmethane pH indicator dyes, varying ring substitution is the means of adjusting the pK of the dye. (5) The phenol red dyed acrylamide is fluorescent (when dry) whereas phenol red alone has at best an extremely weak fluorescence. This fluorescence indicates fixation of the indicator rings, since the classic fluor, fluorescein CI45350, is merely the same type of dye, phenolphthalein, made fluorescent by linking the two indicator rings with an oxygen bridge to prevent their free movement. pKof Bound Dye. The effective pK of the dye in the pH probe can be found with the use of Equation 10. At a sufficiently acid pH, such as A = -4, R - k
(12)
and at a sufficiently basic pH, such as 1 = +3
R
k
(13)
X
Determination of R a t extremes of p H showed a typical probe to have fi = 0.70 and C = 1.45. Substitution of C and k into Equation 10, using Equation 5 along with (R,pH) values in the measurement range, provides an estimate of the pK. This was found to be 7.57 for probes made as described. Measuring Instrument. The details of the measuring instrument are described in a separate publication ( 2 ) . A highintensity tungsten lamp source injects light into the illuminating optical fiber. The light returning from the probe through the other optical fiber is selected into two wavelengths by a cycling filter wheel. The ratio R of the green (560 nm) light intensity, which decreases with increasing pH, to the red light intensity (greater than 600 nm) used as a reference, is determined after subtraction from each of the dark signal in the sensing photodiode. This ratio is then converted to pH by a linear equation pH = gain
X
R
+ level
(14)
where the gain and level are calibration adjustments on the instrument. The output is read directly as pH on a digital meter with a voltage output provided for a recorder. Calibration requires a measurement to be made in two separate buffers, since it has not been possible to obtain a sufficiently reproducible gain factor for the change in p H with R among different probes. For calibrat,ion purposes, the instrument has provision for reading the green to red ratio R directly on the digital meter. Calibration consists of inserting the probe into two different buffers, ordinarily pH 7.0 and pH 7.4, and observing the valne of R in each buffer. Equation 14 is then solved simultaneously for the two sets of ( R ,pH) values to obtain the gain and ievel setting for the instrument. The effect on calibration of a change in ambient light level is negligible. For example, a change
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
867
from normal room illumination to darkness increased the pH indication by 0.005, P E R F O R M A N C E TESTS OF T H E P H P R O B E Response. T o demonstrate the precision and linearity of response of the p H probe, it was compared with a commercial laboratory p H glass electrode in 0.05 M phosphate buffer in a water jacketed beaker with stirring to maintain the temperature at 25.0 "C. The pH probe was calibrated at pH 7.00 in the fresh buffer, and at pH 7.40 by adjusting the buffer with KOH solution. Dilute HC1 or KOH was then added to the buffer to change the p H back and forth in the region of p H 7.0 to 7.4, in steps of about 0.1 pH unit, and the pH readings of the probe and electrode were recorded. The straight line regression of the 11 p H probe values on the glass electrode values gives a line which deviates from the calibration line by a maximum of 0.0075 pH unit, a t pH 7.0. The individual points vary from the calibration line with a standard deviation of less than 0.01 pH unit. The theoretical line from Equation 10 matched to the calibration points provides a slightly better fit with the data points because of a small curvature in the line. These results indicate that the probe meets the design objective of measurements to 0.01 pH unit in the pH 7.0-7.4 range. It should be pointed out that the probe can be used for measurements beyond the range of pH 7.0 to 7.4 with either a sacrifice in the accuracy of measurement or recognition of the curvature which develops beyond this range. Since this probe was intended for physiological use, it was not regarded as necessary to consider its application beyond this narrow p H range. It was of greater interest to design a system which would have the ability to make pH measurements approaching a n accuracy and precision of 0.01 pH unit, as nearly as possible. T e m p e r a t u r e Coefficient. The temperature coefficient of the pH probe was measured by observing the apparent pH reading of a calibrated probe in a buffer solution while varying the temperature of the buffer solution from 20 to 40 O C . The buffer was a 0.05 M phosphate buffer for pH meter calibration at p H 7.00 at 25 "C. The value for the p H of the buffer solution was corrected for its change in buffer pH with temperature in accord with the information furnished on the bottle of buffer (Baker and Adamson). The temperature coefficient, expressed as change of pH probe indication per "C, was 0.0174 over this temperature range. In comparison, the change of the indicated p H of a glass electrode with change in temor -0.0235 a t p H 7 and 25 "C, from the perature is -pH/?', Nernst equation. Ionic S t r e n g t h Coefficient. It was desirable to find the extent to which the p H probe would be affected by ionic strength variations, since dye indicators are susceptible to error from this source. A series of buffers were prepared, using Tris.HC1 and Tris (tris-hydroxymethylaminomethane, Sigma Chemical Co.), containing varying amounts of sodium chloride to vary the ionic strength, and all were adjusted to pH 7.00 with a p H meter calibrated in 0.05 M phosphate buffer. The total Tris concentration was 0.05 M and its contribution to the ionic strength was calculated to be 0.046 M, on the basis of the degree of dissociation of Tris-HC1 a t pH 7.00. Sodium chloride was added to range the ionic strength from 0.05 to 0.3. The indicated p H of a precalibrated probe was obtained in these buffers. T h e p H probe was calibrated as described previously. A straight line regression of the probe values on the log of the ionic strength in eight different solutions showed a change of 0.01 pH unit per 11.0% change in ionic strength. I t is interesting that this is close to the value of 10.5 which is obtained over this ionic strength range from the salt correction given by Bates ( 3 ) ,and the value of 9.3 calculated from the experimental data of Kolthoff given in the same place.
- -0
4
3
2
1
DISTANCE FROM FIBERS TO END OF CELL, rnm
Figure 3. Variation of intensity of returned light with length of probe packing Response T i m e of t h e Probe. As would be expected for a diffusion situation, the probe follows a step change in the pH of the surrounding solution in an exponential manner, with a characteristic time constant of 0.7 min for a (1- ( l / e ) ) , or 63% response. Experiments on probe construction, and calculations based on diffusion coefficients, resulted in the conclusion that response time is inherently characteristic of the dimensions of the probe. Smaller diameter probes might be faster, and were made in early feasibility tests, but they suffered from decreased mechanical strength of the smaller fibers and greater difficulty of construction. Effective Cell P a t h Length. The unique cell construction, in which reflecting particles return the light in the direction from which it comes, does not provide a known path length L. It was of interest to measure the effective light path in the probe so the necessary packing length would be known. An experimental measurement of the path length was made by driving a pair of optical fibers into a well in a glass plate, filled with buffered pH probe packing. The well was a length of 1-mm i.d. glass capillary tubing, approximately 2 mm long, cemented to a microscope slide. This was mounted on a Harvard infusion pump syringe drive together with the fiber pair, so that they traveled into the well and ultimately reached the end of the well with the fibers in direct contact with the glass slide. The intensity of the light signal passing in one fiber and back out the other fiber was charted on a recorder synchronized with the movement of the fibers into the well. This showed that a length of 2 mm for the probe packing was more than sufficient, and most of the absorption of the light by the dye occurred in a few tenths of a millimeter. Figure 3 shows the extent to which the intensity of returned light decreases as the length of the packing is shortened. Stability of t h e Probe. Although the probe was intended to be of inexpensive disposable construction, it can be used, dried out, and reused repeatedly as long as it is not mechanically damaged. It can be used physiologically for at least a few hours. It can be left in a solution for several days. The drift in calibration of the probe is less than 0.01 p H unit in 2 h. If it is dried out, it must be recalibrated before reuse. I n - V i t r o Blood Evaluation. A similar test of the probe was made to evaluate its performance in blood. A comparison calibration of the probe with a pH electrode was done as above. The electrode and probe were then immersed in heparinized dog blood in a water-jacketed stirred beaker a t 25.0 "C. The pH was changed from the original 7.53 pH electrode reading of the dog blood to p H 7.0 in steps of about 0.1 p H unit by addition of dilute HC1, and back again to pH 7.425 with
868
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980
'
7m5 I
Respirator 1 Limin 0 2 2% Halothane
-
___-___---------
*Off respirator. breathing room air
\
I I
'\
7.4
-
C 0 2 % L/rnin
1 Llrnin
e a
7.3 -
PH
7.2
I
7.1
-
7.0
1
6.9
'
0
25
50
75
1
1
I
I
100
125
150
175
J
MINUTES AFTER INSERTION Figure 4. In-vivo evaluation of pH probe in sheep
addition of dilute KOH, giving 10 values of the fiber optic probe compared with the glass electrode. The straight line regression of the p H probe values on the pH glass electrode values matches the straight line drawn between the buffer calibration points, with a maximum difference of 0.007 pH unit, and the points are within 0.017 p H unit standard deviation from the calibration line, provided that the two points a t p H greater than 7.4 are omitted from the calculations. These two points (pH 7.53 and 7.425 with the glass electrode) indicate a strong curvature of the relation beyond p H 7.4 in the blood, with the fiber optic probe values falling below the electrode values. The straight portion of a theoretical curve based on Equation 10 also matches the calibration line. The theoretical curve is based upon the aforementioned pK = 7.57, and values of k = 0.797 and C = 0.956 for this probe, derived from fitting Equation 10 to the two calibration points and solving simultaneously for k and C. In-Vivo Blood Evaluation. The fiber optic probe was tested in an animal to evaluate its suitability for in-vivo blood p H measurements, in comparison with a pH electrode and with p H measurements on blood samples determined with a laboratory p H meter. The fiber optic pH probe was calibrated at 39 "C in buffers, with reference to electrometric pH, with appropriate temperature correction. The probe, with a Teflon jacket over the fibers, was inserted through an 18-gauge needle (0.825-mm i.d.) into the right jugular vein of an anesthetized 61-kg adult ewe sheep (Dorset-Rambouillet cross). A pH microelectrode (model MI-502, Microelectrodes, Inc., Grenier Industrial Village. Londonderry, N.H. 03053) was inserted into the left jugular vein through a 14-gauge needle (1.60-mm i.d.) after calibration in the same buffers. A 3-way blood sampling stopcock connected to an intravenous catheter was inserted into the right jugular vein near the fiber optic probe. Blood samples withdrawn from this by syringe were measured on an Instrumentation Laboratories Model 231 blood gas analyzer
calibrated with Instrumentation Laboratories buffers. T h e animal was given a 30-mg dose of heparin anticoagulant at the beginning of the experiment. Respiration was controlled with a Harvard Respiration Pump adjusted to 12 strokes/min with a 700-cm3 stroke volume, connected to the animal with a cuffed endotracheal tube. The respirator pump was connected in the circuit of a Foregger Foretrend Model 100 anesthesia machine, with a 1 L/min oxygen flow input and 2% Halothane anesthetic, except as described below. Carbon dioxide was connected to the nitrous oxide hospital service connection of the anesthesia machine, and the flow was regulated as noted below. The test was carried out by varying the blood pH by control of the C02level in the respiration gas mixture. Figure 4 shows the results of the experiment. For the first 40 min after insertion of the test devices, the animal was maintained without COz. Starting at 40 min, COS was added a t the rate of 0.5 L/min, thereby representing 1/3 of the respiratory gas mixture. The pH decreased continuously until, from 71 to 76 min, the endotracheal tube cuff failed, and the tube was replaced, during which period the animal breathed ambient air. This caused a precipitous rise in pH, followed by a continuous decrease while the COz was maintained as before up to 103 min. After stopping the C 0 2 at this point, the p H rose, and between 116 min and 122 min the animal was intentionally allowed to breathe room air to cause a more rapid pH increase. After reconnecting the animal to the respirator, the pH resumed its drop. To cause a more rapid decrease in pH, the C 0 2 was reinstituted at the rate of 1 L/min (50% of the respiratory gas mixture) between 130 and 138 min, during which time the p H dropped very rapidly. At this point the Halothane concentration was increased to 5% to overanesthetize the animal so that the period off the respirator, with the animal breathing room air, which follows at 141 min, would last longer than before. At 141 min, heart beat irregularity began to appear due to the excess Halothane, and the animal
Anal. Chem. 1980, 52, 869-875
was disconnected from the respirator pump. The pH rose very rapidly while the animal breathed room air. At 152 min, the animal began to wake u p and was reconnected to the respirator. T h e experiment was ended at 165 min. Following the initial insertion, the probe and electrode drifted in opposite directions, and then throughout the experiment the fiber optic probe tended to give lower p H readings than the electrode. During the last half of the experiment, the fiber optic probe showed a faster response to p H change than the electrode did because, as was shown when the probe and electrode were withdrawn a t the end of the experiment, the p H electrode had a heavy coating of adherent biological material covering it, while the fiber optic probe had a barely discernible coating. T h e p H values determined on blood samples by the laboratory blood gas analyzer were in as good agreement with the fiber optic probe as with the electrode. It is not possible to say which of the measurements is most correct. This demonstrates that the fiber optic probe is generally useful for blood p H measurements in-vivo, and gives as good an indication of the p H level as electrode methods.
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ACKNOWLEDGMENT We would like to express our thanks to the National Heart, Lung, and Blood Institute, and Ronald Crystal and Robert M. Winslow of that Institute, for financial support of this project. Also, we thank Joseph E. Pierce of the Institute for providing the source of sheep and arranging the use of the Institute's surgical laboratory.
LITERATURE CTTED (1) Cbrk, W. Mansfield. "The Determination of Hydrogen Ions"; Williams and Wilkins: Baltimore. Md., 1928; Chap!ers 5 , 6, and 7 . (2) Goldstein, S.; Peterson, J. 1.: Fitzgerald, R. V. ASME Trans. J . Bbmech. Eng., in press. (3) Bates, R. G. "Determination of pH"; Wilsy: New York, 1964; p 147.
RECEIVED for review October 29, 1979. Accepted January 18, 1980. The construction of the device described herein and the dye copolymerization are the subjects of {Jnited States and foreign patents. Inquiries concerning use and development may be directed to the United States Department of Commerce, National Technical Information Senice, 5285 Port Royal Road, Springfield, Va. 22161.
Separation of Metal Salts by Insolubilized Noncyclic Poly(oxyet hy lene) Derivatives Hiroshi Fujita, Shozo Yanagida, * and Mitsuo Okahara Department of Applied Chemistry, Faculty of Engineering, Osaka University, Yamadakami, Suita, Osaka, Japan 565
The selectivity of cation binding of insolubilized noncyclic poly(oxyethy1ene) derivatives (POE) is described. The POE resin beads are synthesized from chloromethylated crosslinked poly(styrene) and poly(oxyethylene) derivatives having various oxyethylene units. Their distribution coefficients and separatlon factors for alkali and alkaline earth metal salts were determined In acetone, 10 wt YO H,O-acetone, and methanol. The POE beads with more than 5 oxyethylene units are highly selective for potassium ion in the latter two solvents. The separatlon of LiSCN, NaSCN, and KSCN was successfully performed by the technique of high-performance liquid chromatography using the POE beads with 7 or 10 oxyethylene units. Their metal salts selectivity depends not only on metal cations but also on counteranions.
Recently several reports on the separation of metal salts by resins with neutral ligands have appeared. Some insolubilized macrocyclic polyethers were successfully used to separate some alkali and alkaline-earth metals salts, and some ammonium salts (1-3). Furthermore, the separation of metal cations was extended to noncyclic ligands insolubilized on the polymer supports ( 4 ) . These functional resins, however, were not necessarily satisfactory because of a multistage preparative requirement. We recently clarified that noncyclic poly(oxyethy1ene) (POE) derivatives are capable of complexing alkali and alkaline-earth metal cations, even if they have no special chelating groups a t the terminal positions (5-7). A series of their solid complexes with alkali and alkaline-earth metal salts were successfully isolated (7) and some of their crystal structures 0003-2700~80i0352-0869$01.OO/O
were clarified by X-ray crystallographic analysis (8). Based on these facts, we proposed that the fulfilment of the energetically favorable coordination geometry of the metal cations and the compatibility of the POI? ligands should be the important factors governing their specific and selective complexation (7). It was further found in the application t o phase-transfer catalysis that their catalysis depends not only on the number of oxyethylene (EO) units b u t also on metal cations (9-11). Taking these facts into account, it became interesting t o study whether metal salts are separable using insoluble poly(styrene)-bound noncyclic POE derivatives. In this paper, the extraction abilities, the distribution coefficients, and the separation factors of some insoluble POE beads were determined by a batch extraction method. T h e practical separation of alkali and alkaline-earth metal salts was achieved by the technique of high-performance liquid chromatography.
EXPERIMENTAT, Materials. The following chemicals were the purest available grades (source): NaSCN, NaI3r (guaranteed reagent (G.R.) grade), LiSCN.2H20. Mg(SCN)2.4H20(extra pure (E.P.) grade)(Nakarai Chemicals, Ltd.); KBr, NaC1, KCl, Ra(SCN)2.2H20,Ca(SCN)2.3H20(practical grade), KSCN, KI (G.R. grade)(Wako Pure Chemicals Ltd.); NaI (E.P. grade) (Kishida Chemicals, Ltd.). Decaethylene glycol (E010) was prepared from the sodium salts of triethylene glycol (E03) and the dichloride of tetraethylene glycol (E04). Tetra-, penta-, hepta-, and decaet hylene glycol monomethyl ethers (MeE04 [125-130 OC/3.0-3.5 mmHg], MeE05 [190-194 OC/7.8-8.2 mmHg]. MeE07 [160-162 0C/0.12-0.14 mmHg), and MeEO10) were prepared from the sodium salt of E 0 1 and E02 chloroethylmethyl ether. from the sodium salt of E02 and E02 chloroethylmethyl ether, from the sodium salt of E04 and E02 chloroethylmethyl ether, and from the sodium salt 1980 American Chemical Society
