Anal. Chem. 1088, 60, 489-493
489
Performance of an Improved Monodisperse Aerosol Generation Interface for Liquid Chromatography/Mass Spectrometry Paul C. Winkler,' Deborah D. Perkins, William K.Williams, and Richard F. Browner* School of Chemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400
An improved monodbpertm aeroroi generatlon Interface for ilquld chromatography/masr spectrometry interfacing (MAGIC-LC/MS) is deacrlbed. The interface has an aerodynamically superlor momentum separator, whlch results in decreased anaiyte loss in passing through the Interface. The interface is shown to perform well with a quadrupole mass spectrometer, in addition to earlier studies with a magnetic sector Inshrment. A new method of formkrg aerosol has been developed, whkh reduces the dead volume dgnlfkantly over earlier designs. The performance of the interface has been evaluated by studying Its capabilities for (1) generating electron impact spectra of searchable quality for selected compounds of Interest, (2) operating wtth typkai llquid chromatographic separation conditions, including reverse phase and gradlent dutlon, and (3) provrcNng low detection lhrlts for both full scan and selective ion monitoring detection of a range of compounds. Studies Include identification of the components of a mixture of CISand trans isomers of the thermally labile compound retinol (vitamin A) acetate. Full scan ( m / z 80-350) electron impact spectra were readily obtained wtth SO-ng tnJectlonsorrcolmn. Detection limits for this compound were 10 ng full scan and 1 ng wlth selected ion monitoring. Identtflcationof a free (nonderlvatized) fatty acid mixture was also readlly obtalned, uslng a reversedphase separation in gradient mode.
The inability of gas chromatography/mass spectrometry (GC/MS) to analyze many involatile and/or thermally labile samples has led to a continuing interest in the development of liquid chromatography/mass spectrometry (LC/MS) as a viable analytical technique. The three most successful approaches to date have been direct liquid introduction (DLI), moving belt (MB), and thermospray (TSP) LC/MS. However, each of these techniques suffers from certain limitations, and as a consequence no one approach is suitable for handling all problems. The DLI interface, for instance, generates only chemical ionization (CI) spectra (1) and is therefore of little use for the analysis of unknown mixtures. The moving belt interface is capable of generating electron ionization (EI) spectra, in addition to CI spectra, but is limited to compounds with a relatively narrow volatility range. Compounds of low volatility are difficult to remove from the belt, while highly volatile compounds may be lost from the belt before entering the ion source region of the mass spectrometer (2). Thermospray (3)provides a combined sample introduction and soft ionization technique that gives molecular weight information but substantially less structural information than that provided by E1 spectra. This limits the usefulness of TSP for the analysis of unknown samples. In addition, thermospray performance varies significantly with sample type, temperature, and buffer concentration (4,5). As a consequence,while 'Present address: Dow Chemical Western Division, Pittaburg, CA
94565.
0003-2700/88/0380-0489$01.50/0
TSP provides a valuable means for LC/MS interfacing there is still a need for an interface system providing reproducible, searchable mass spectra. Such spectra should be of a predictable type, ideally approximating the reference E1 spectra available on databases. The capability for production of standard CI spectra is also desirable. Recent work in our laboratory has focused on the developoment of a monodisperse aerosol generation interface for combining liquid chromatography with mass spectrometry (MAGIC-LC/MS). The MAGIC interface has been specifically designed to overcome a number of the functional problems experienced with other LC/MS approaches. MAGICLC/MS can be used to generate either E1 and/or CI spectra of compounds that are either too involatile or too thermally labile to allow their direct analysis by GC/MS. While early designs of the interface have proved successful in generating E1 spectra of involatile compounds and in producing detection limits in the nanogram range, a number of features were in need of improvement. Specific problems with the previous model of the interface (6) were (1)difficulty of maintaining accurate nozzle/skimmer alignment, (2) poor reproducibility, (3) inadequate sensitivity for many separations, and (4) undesirably large dead volumes in both the aerosol generator and and aerosol evaporation chamber. This paper describes the construction and preliminary evaluation of an improved MAGIC-LC/MS interface, which overcomes many of the limitations of earlier models. EXPERIMENTAL SECTION Instrumentation. The mass spectra were recorded by using a Hewlett-Packard5988A quadrupole mass spectrtometer. The electron beam was operated at 70 eV. Source temperature was 240 O C , unless otherwise stated. Mechanical pumps used for the interface were Duo 16B two-stage rotary pumps (Balzers, N. Arlington Heights, IL), with 21.6 m3/h capacity. These pumps are designed specifically to cope with high solvent loadings without the need for cyrogenic trapping. The liquid chromatography was carried out with a HewlettPackard 1090A binary gradient system liquid chromatograph. Columns used were (1)reverse phase, 2.1 X 100 mm Hypersil ODS reversed-phase column packed with 5-rm particles (HewlettPackard) and (2) normal phase, 4.5 x 250 mm column packed with 5-rm silica particles (Altech). Reagents. All solvents were Fisher reagent grade quality and were fdtered and degassed before use. The retinol acetate isomeric mixture was provided by Dr. B. Fair, and pure trans-retinolacetate was obtained from Sigma Scientific (St. Louis, MO). The fatty acids were obtained from Sherex (Dublin, OH) and Universal Preserv-a-Chem (Brooklyn, NY). All chemicals were used as received.
RESULTS AND DISCUSSION The MAGIC Interface. The basic concepts of the MAGIC interface have been discussed in detail elsewhere (6, 7). The effluent from an HPLC column is pumped through a small (typically 25-pm-diameter) orifice resulting in the formation of a liquid jet. the liquid jet spontaneously breaks up through Rayleigh interactions with the surrounding gas into uniform drops whose diameters are approximately twice that of the of the initial liquid jet. The stream of closely spaced drops Q I988 Amerlcan Chemlcal Soclety
490
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988
is then subjected to a perpendicular flow of helium gas which initially disperses and prevents agglomeration. By use of a higher dispersing gas velocity than in previous work ( 6 ) ,the primary drop stream is then subjected to a secondary shearing force, which reduces the mean drop size of the aerosol and so improves the solvent vaporization rate in the evaporation chamber. The aerosol is then directed through a glass chamber maintained at a pressure slightly below ambient, where solvent evaporates rapidly from the drops. As solvent evaporates, any analyte present in the drops forms a solid residue, thus becoming a high velocity particle beam directed at the momentum separator portion of the interface. If the analyte present in the original drops should be liquid rather than solid at room temperature, then the desolvated beam will be composed of liquid drops, rather than of solid partticles. The desolvation chamber is connected to the momentum separator. On leaving the evaporation chamber the mixture of He dispersal gas, solvent vapor and analyte particles expands through a nozzle, undergoing supersonic expansion into the first low-pressure region (approximately 10 Torr). This results in a high-velocity gas jet containing suspended analyte particles. The high mass of the analyte particles relative to the gas stream provides them with much greater momentum than the solvent vapor molecules and He atoms. Consequently, the particles do not expand from the core of the expansion jet as rapidly as do the gases. Skimming the core of the expansion jet therefore results in substantial enrichment of analyte relative to He and solvent vapor. The solvent vapor that has been skimmed off then is pumped away mechanically. This process is repeated in the second stage of the momentum separator, dropping the chamber pressure to approximately 1Torr. After leaving the momentum separator, the analyte particles continue on a linear trajectory into the ion source region of the mass spectrometer. The narrow particle beam (z2-mm diameter) enters the ion source area of the mass spectrometer, where it undergoes a very rapid process of flash vaporization. This results in the production of a high transient vapor pressure consisting largely of intact analyte molecules. The mass spectra of these molecules generally show little evidence of thermal degradation. The vapor-phase analyte molecules are then ionized, using either electron impact or chemical ionization processes as desired, and the mass spectra measured in the normal way. Effective operation of the interface both with magnetic sector (e.g. Finnigan-MAT 112s) and quadrupole mass spectrometers (e.g. H-P Models 5930A and 59f58A), has been achieved without the need to make any changes in either the configurations, pumping capabilities, or geometries of the mass spectrometers used. Aerosol Generator. In the original version of the aerosol generator (6),the liquid jet was formed through a drawn-out glass capillary tube. The conical bore of this tip caused frequent clogging in the presence of fine particles. Blockage resulted from either Teflon or glass particles collecting in the jet tip. These particles proved difficult to remove by either ultrasonic or other cleaning procedures. Additionally, the tip possessed a relatively large dead volume, which contributed to significant peak broadening. The dead volume arose primarily from an inefficient seal between the glass aerosol generator tip and the LC tubing outlet from the chromatographic column, which proved very difficult to eliminate. Finally, the diameter of the orifice was difficult to reproduce in the construction of several tips with the same nominal orifice size, due to difficulty in controlling the hand grinding process used in their fabrication. The newly designed aerosol generator (Figure 1) uses cylindrical bore fused-silicacapillary tubing in place of the conical glass tip of the earlier aerosol generator. The fused-silica capillary is of the type used routinely for capillary GC ap-
ALUMINUM BODY
\ 220pm DIAMETER TUBING FROM HPLC
Figure 1. Cross
25 urn TUBING FOR LIQUID JET
TUBING CONNECTOR
sectional view of new MAGIC aerosol generator.
plications, and has a polyimmide coating. In the new aerosol generator design, a short length of 25-pm-i.d. fused-silica tubing is connected to a length of 220-pm4.d. fused-silica tubing using a zero dead volume fused-silicaconnector (Valco). The 220-pm capillary tube is used to connect the aerosol generator with the LC column. The liquid jet is formed at the tip of the 25-pm4.d. tube. This aerosol generator is much simpler than the previous design, and seldom clogs. When clogging does occur, the small-bore fused-silica tube may be replaced in 2-3 min. With the use of identical solvent flow rates and injection volumes, the peak width of an injection, without a column in line, is approximately 6 s wide for the new aerosol generator, compared to approximately 24 s wide with earlier aerosol generator. This represents an improvement of 4X. The peak shapes observed with the new aerosol generator also show none of the peak tailing that was often observed with the old design of aerosol generator. Momentum Separator. Transport efficiency studies have indicated that analyte loss in the MAGIC interface occurs primarily in the momentum separator. The most important analtye loss processes are the result of particle sedimentation, poor nozzle/skimmer alignment, and turbulence loss (8,9). The earlier MAGIC interface (6) incorporated two cylindrical-bore tubes in the nozzle/skimmer design, with each tube capable of adjustment in the r-y plane at one end only. This approach suffered from two major disadvantages. First, accurate alignment was difficult, because only one end of the tube could be moved during the adjustment process. This tended to cause the axial alignment of the skimmer and nozzle tubes to be disturbed during adjustment. Second, the tubes were typically several centimeters long, which resulted in substantial particle loss due to particle impaction from the expanding beam onto the tube walls. The nozzles and skimmers used in the original MAGIC interface were made from thick-walled glass capillary tubing. In such a system the skimmers have flat faces perpendicular to the direction of the gas flow, and possess a large lengthto-diameter ratio. This type of skimmer significantly disturbs the gas flow in the skimmer vicinity, resulting in turbulence (10). When an injection is made of an analyte in relatively high concentration, the diameters of the particles left after solvent evaporation are also relatively large. When the injected analyte has a lower concentration, the diameters of the desolvated particles will be proportionally reduced. These smaller particles will be more subject to loss by turbulent processes, as they will more readily follow the path of the gas flow. In the present work, a prime goal was to improve the transport efficiency of the interface, and hence to improve detection limits. Consequently the design of the new skimmers was aimed at providing an undisturbed path for the particle beam through the momentum separator. The aerodynamic principles discussed by Kantrowitz and Grey (10) served as a guide for this work. A schematic of the improved MAGIC momentum separator is shown in Figure 2. The new design is substantially shorter than the original version, and the nozzle and skimmers are fixed in place radially for proper alignment. The momentum separator body is made from stainless steel. Nozzles and
ANALYTICAL CHEMISTRY, VOL. BO, NO. 5, MARCH 1, 1988
491
PUMP PORTS
5 W
328
W KO io0
300
200
miz NOZZLE
SKIMMERS
#2
105
Figure 2. Cross sectlonai view of new MAGIC momentum separator. m w 328 #2
/I 300
200
io0
mlz Figure 4. Mass spectra of CIS- and trans-retinol acetate. 2 ng
Figure 3. Total ion chromatogram of retinol acetate mixture. Conditions: 9 5 5 hexane/ether solvent at 1 mL/mln on 4.5-mmi.d. column, packed with 5-pm silica particles.
skimmers are machined from 6010 grade aluminum. The nozzle has a 0.5-mm orifice at the apex of a 60° cone. The separation between the nozzle and first skimmer is continuously variable from 0 to 10 mm through the use of shims. Vacuum seals between the various chambers are with Viton O-rings. The first skimmer has a looo exterior angle and a 95O interior angle, with a 0.5-mm orifice at the tip. The second skimmer has a 45O exterior angle and a 30° interior angle, with a 1.0-mm orifice. The separation between the skimmers is adjustable from 0 to 10 mm. Both vacuum chambers are pumped with mechanical vane (hot oil) pumps of 21.6 m3/h capacity. A tubular vacuum inlet, with the same dimensions as a direct inlet probe (DIP) or standard GC interface probe, is bolted directly to the second skimmer. This allows the interface to be connected rapidly and simply to the mass spectrometer directly through the DIP vacuum lock. Accurate alignment of the probe to the ion source is then provided by locating the probe tip against the ion source inlet. The improved design has many advantages over the previous design: (1)it is easier to set up and operate, resulting in less down time of the mass spectrometer, (2) the alignment between nozzle and skimmers is fixed and its accuracy depends only on the quality of the component machining, (3) the skimmer design is aerodynamically superior, resulting in a less turbulent gas flow through the interface, (4) the new momentum separator has higher transport efficiency than the previous design, which results in improved detection limits, and (5) the lower dead volume in the aerosol evaporation chamber causes less peak broadening, which improves both interface chromatographic resolution and analyte detection limits. Performance of the Improved Interface. Normal Phase Separation. of Retinol Acetate Isomers. The separation of a mixture of cis- and trans-retinol acetate (50-ng injection) is shown in Figure 3. The total ion chromatogram was not smoothed in any way and is typical of the low noise level
z
.
0 0
1
2
3
TIME (min.)
Flgure 5. Single Ion monitoring trace of trans-retinol acetate at m l z 119.
obtained with the MAGIC-LC/MS interface. A column of 4.5-mm i.d., packed with 5-pm silica was used. Elution was isocratic, with a 95:5 hexane-ether mixture at 1 mL/min. Attempts to isolate these isomers after a preparative scale separation resulted in decomposition because of their high thermal lability. With the MAGIC interface it was readily possible to separate and identify the mixture components on-line. The mass spectra are shown in Figure 4. The spectra show a clear molecular ion at m / z 328, indicating that little thermal degradation has occurred and that the fragmentation pattern is consistent with the molecular structure. The spectra were compared to an NIH reference spectrum (11) of unresolved retinol acetate isomers and were found to compare favorably. The quality of the E1 mass spectra generated were sufficiently high to have allowed identification of the compounds in the mixture had it been an unknown. It should be noted that the mass spectra shown in Figure 4 provide useful information down to m/z 80,the lower limit of the scan. However, this is not the lower limit of the scan range possible with the interface, which is set only by m/z values of ions formed from the chromatographic solvent species. With typical LC solvents (e.g. H20, MeOH, CH3CN), scans down to m/z values in the range 19-41 are routinely possible, allowing ready access to low-mass fragments as an aid to compound identification. This represents a significant advantage over Thermospray LC/MS, which produces high background levels of ions with m/z values up to 100. As a consequence, Thermospray gives little information regarding structurally significant low-mass ions.
492
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988
,
#1
100
+ W
1
#2 1 4
CH,
C.,
z
+
CH.
C"
50
mw228
FH.
CH.
4
CH'
w
CH'
n
CH, CH,
0
2
0
4
S
6
10
TIME (min)
Flgure 6. Total ion chromatogram of mixture of fatty acids: 1, lauric acid; 2, myristic acid; 3, palmitic acid; 4, stearic acM. Scan 80-450. Conditions: ODS reversed-phase column (2.1 X 100 mm); gradient from 80:20 MeOH/H,O, with 3 % acetic acid, to 100% MeOH.
~
100
150
IS?
#3
loo
I1 I,' I 0 U III'l
100
I
250
Figure 8. Electron impact mass spectrum of peak 2, myristic acid, molecular weight 228.
\
Ill1
200 mlz
i
.&I,
150
I I / .
200
3
si29
is I I
.
Cn,
.
250
rnlz
Electron impact mass spectrum of peak 1, lauric acid, molecular weight 200.
Figure 7.
0
A single ion monitoring trace of 1- and 2-ng injections of retinol acetate (trans isomer) at m / t 119 are shown in Figure 5. The E1 response of retinol acetate is poorer than for many compounds that generate E1 spectra in the MAGIC-LC/MS interface, but the data are certainly indicative of what may be routinely achieved when using the interface without any special optimization strategy. Lower detection limits are possible for other compound types. Reversed-Phase Separation of Fatty Acids. For an LC/MS interface to be of broad value for the routine identification of unknown mixtures, it must be capable of generating library searchable E1 spectra of compounds not readily amenable to GC/MS. Many of these separations will also require reverse-phase chromatography with gradient elution. Ideally, the interface should be able to operate equally well under gradient conditions as it does isocratically and furthermore to handle solutions with a high percentage of water. It should not be necessary to derivatize ionic compounds to enhance volatility prior to introduction to the interface, as this negates one important advantage of LC over GC. The classes of compounds most suitable for making this comparison are those which are thermally labile and/or relatively involatile. For this reason, a series of fatty acids, lauric (CH3(CHz)&OOH) through stearic (CH3(CHz)&OOH) were selected. Separation of these compounds is not possible directly by GC/MS, but requires derivatization. A total ion chromatogram for a mixture of lauric, myristic, palmitic, and stearic acids (500-ng injection) is shown in Figure 6. The separation was on an ODS reversed-phase column (2.1 X 100 mm), using a gradient from 80:20 MeOH/H20,with 3% acetic acid, to 100% MeOH in 10 min, a t a flow rate of 0.5 mL/min. The scan was from 80 to 450, and the source temperature was maintained a t 240 OC. The E1 spectra of the peaks are presented in Figures 7-10. The E1 spectra show good molecular ions and are in excellent agreement with the
100
150
200
250
miz
Electron impact mass spectrum of peak 3, palmitic acid, molecular weight 256.
Flgwe 9,
#4
100
17
100
I
cn,
$;:
Ll
I
150
200
250
mlz
Figure 10. Electron impact mass spectrum of peak 4, stearic acid, molecular weight 284.
EPA/NIH reference data base (11). Clearly, there is little evidence of thermal degradation occurring in the interface, in spite of the relatively high temperature of the ion source (240 "C). This suggests that rapid flash vaporization of the analyte species occurs when the dry particle beam enters the ion source area, and that the time scale is sufficiently rapid for minimal thermal degradation to occur. The lack of serious thermal degradation in the source observed with MAGIC-LC/MS has also allowed the generation of searchable E1 mass spectra from a number of environ-
493
Anal. Chem. lS00, 60, 493-490
mentally important, and thermally labile compounds such as Aldicarb, Silvex, and Cyanazine. Detailed studies with these compounds will be reported elsewhere. It should be noted that all the E1 mass spectra illustrated in this paper (Figures 4 and 7-10) are without background subtraction. The low background levels shown are typical for normal MAGIC-LC/MS operation. This is in contrast to the high chemical noise which is found with the moving belt interface, resulting from continuous thermal desorption of phthalate plasticizer from the belt material. Scope of the MAGIC-LC/MS Interface. As yet, the full range of compound types accessible with the MAGIC-LC/MS interface has not been fully investigated. In general terms, all compounds that are known to be capable of generating E1 and CI spectra and that have been tried with the interface have generated good quality spectra Thew include carbamate and triazine pesticides, phenyl urea herbicides, polynuclear aromatic hydrocarbons, plant alkaloids, antioxidanta, and EPA Appendix 8 compounds. Compounds known not to generate E1 spectra, such as simple sugars and certain azo dyes, predictably do not generate spectra with the current system. The primary mode of ion formation appears to be through a flash vaporization step in the ion source, followed by E1 or CI ionization, as selected. Any limitation on compound type accessible with MAGIC-LC/MS will be influenced by (1) whether the molecule is functionally capable of generating an E1 spectrum and (2) whether the compound is sufficiently volatile to be capable of forming an adequate vapor pressure in the ion source. This latter property will ultimately represent the interface limitation for generation of E1 and CI spectra,
rather than the molecular weight of the species. Nevertheless, it is possible to generate good searchable E1 spectra with strong molecular ions even for quite involatile species. An example is provided by reserpine, with a molecular weight of 608, which generates a searchable E1 spectrum with a strong molecular ion at m / z 608. Registry No. &-Retinol acetate, 29443-87-6; trans-retinol acetate, 127-47-9;lauric acid, 143-07-7;myristic acid, 544-63-8; palmitic acid, 57-10-3; stearic acid, 57-11-4. LITERATURE CITED (1) (2) (3) (4) (5) (6) (7)
Arpino, P. J. J. Chromefogr. 1985, 323, 3. Krost, K. J. Anal. Chem. 1985, 5 7 , 763. Vestal, M. L. Int. J . Mass Spectrom. Ion W y s . 1983, 46, 193. Blakley, C. R.; Vestal, M. L. Anal. Chem. 1963, 55, 750. Voyksner, R. D.; Yinon, J. J. Chromafogr. 1966, 354, 393. Willoughby. R. C.; Browner, R. F. Anal. Chem. 1984, 5 6 , 2626. Browner, R. F.; Winkler, P. C.; Perklns, D. D.; Abbey, L. E. Mlcrochem. J . 1966, 3 4 , 15. (8) Browner, R. F.; Boorn, A. W.; Smith, D. D. Anal. Chem. 1962, 54. 1411. (9) Hinds, W. C. Aerosol Technology; Wlley-Interscience: New York, 1982. (IO) Kantrowitz. A.; Grey, J. Rev. Sci. Insfrum. 1951, 22, 328. (11) EPAlNIH Mass Spectral Data Base; Heller, S . R., Milne, G. W. A.; Eds.; U.S. Government Printing Office: Washington, DC, 1978.
RECEIVED for review September 16, 1986. Resubmitted October 29,1987. Accepted November 16,1987. This research was supported by the U S . Department of Energy, Office of Basic Energy Sciences, under Grant No. DE-FG05-85E13435. We are grateful to Hewlett-Packard Scientific Instruments Division, Palo Alto, CA, for the loan of the 5988A mass spectrometer.
Elimination of Neutral Species Interference at the Ion-Sensitive Membrane/Semiconductor Device Interface Xizhong Li, Elisabeth M. J. Verpoorte, and D. Jed Harrison*
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T 6 G 2G2
Semlconductor electrodes coated with a K+-responslve pdy(vlnyi chiorlde) (PVC)-based membrane, wlth the strucare found to respond to K+ ture n-SI/SlO,/SI,N,/membrane, In a Nernstlan fashion vla a fleld-effect mechanism. Such electrodes respond to CO,, benrolc acid, and ascorbic acld as Interfered. However, lntroductlon of a Ag/AgCI layer to glve n-SI/SIO,/SI,N,/Ag/AgCl/membrane electrodes ellmlnates the interference. Spectroscopic evidence clearly shows that benrolc acld permeates the membrane at low pH. Analysts of the Impedance response of PVCbased K+ membranes demonstrates that all the lnterferents permeate the membrane, resultlng In a reversible reductlon In bulk membrane resistance. Relevance to Ion-sensltlve field-effect transistor devices is discussed.
Ion-responsivesemiconductor devices such as the field-effect coupled diode or ion-sensitive field-effect transistor (ISFET) are the subject of continued research interest (1-3). Recently, it has been reported that pH-sensitive ISFETs coated with poly(viny1 chloride) (PVC)-based K+-sensitivemembranes are subject to a number of unexpected interferences (4). Species 0003-2700/88/0360-0493$0 1.50/0
such as COz, acetic acid, an benzoic acid, which show no interference at ion-selective electrodes (ISE) employing the same membrane, cause significant potential shifts at K+ membrane coated ISFET's (4). Diffusion of neutral species through the membrane to the silicon nitride surface of the device gate, resulting in a pH shift at the gate, is postulated as the cause of this effect (4). In ISFET operation, the potential induced via a field effect in the Si substrate is controlled by the charge state of the silicon nitride/membrane interface (1-3). For fixed K+ concentration in solution the surface silicon nitride charge will not remain fmed if the proton activity varies at the solid surface. Consequently, the standard cell potential of the ISFET/reference electrode system will be dependent on the history of the silicon nitride surface if the membrane is permeable to acids or bases. Solid-state contacts to replace the internal reference solution of a K+-sensitivePVC, dioctyl adipate, valinomycin ISE have been previously proposed (5,6).The most successful solidstate internal contact is the Ag/AgCl reference system. Simon has pointed out (7) that by incorporating KB(C,H,), in the PVC membrane, in addition to the other components, the Ag/AgCl/membrane/solutionstructure is unblocked to charge transfer at each interface and the thermodynamics are not 0 1988 American Chemical Society
