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Anal. Chem. 1980, 52, 1929-1931
Piezoelectric Crystals as Detectors in Liquid Chromatography Pamela L. Konash Department of Chemistry, Georgetown University, Washington, D.C. 20057
Glenn J. Bastiaans” Department of Chemistry, Texas A& M University, College Station, Texas 77843
Preliminary work is reported on the application of piezoelectric crystals as mass detectors in liquid chromatography. Two of the three major problems associated with extending piezoelectric adsorbed mass detection to the liquid phase have been overcome. Both stable oscillation at a liquid interface and the detection of surface adsorption excluslve of density effects have been achieved. However, this type of LC detector still compares poorly with respect to the other modes of LC detection because methods of enhancing and controlling surface adsorption have not yet been found. Nevertheless, the promising preliminary results presented here indicate that a potential for developing a new sensttive detector for LC exists.
High-performance liquid chromatography (HPLC) as a method of analysis has shown great advancements in theory, technique, and applicability in the past 10 years. T h e successes of modern H P L C are mainly due t o improvements in instrumental design. The development of improved injection ports, pumps, and columns has led to efficient separations of nonvolatile and high molecular weight compounds which are not readily isolated with gas chromatographic (GC) methods. However, one of the weakest links of H P L C systems is the detector. Presently the most widely used detection systems in HPLC are t h e refractive index detector (RI), the ultraviolet adsorption and fluorescence detectors (UV), and the amperometric electrochemical detector (EC) ( 1 , 2 ) . The RI detector measures a bulk property of the solute-solvent system and may thus be considered a universal detector. However, refractive index variations in the solvent cause the RI detection system to suffer from poor sensitivity of detection. The UV and EC methods are more specific because they require solutes t o exhibit specific properties, either light absorbance in the UV spectral region or electrochemical activity, respectively. This specificity may be advantageous if the separated compounds of interest exhibit the appropriate properties. If the solvent system exhibits similar properties which interfere with detection or if the compounds of interest do not display optical or electrochemical activity under the detector conditions, then one is left without a sensitive method of detection. Thus, improvements in H P L C detection systems may be expected t o produce further advances in the utility of this analytical method. One interesting, although not widely used, approach to detection in gas chromatography has been the use of piezoelectric crystals to detect the adsorbance of solute present in the gas phase onto the crystal surfaces (3-5). While the extension of this approach to the detection of solutes in a liquid phase may initially seem quite natural and logical, several problems must be overcome to actually accomplish such an extension. Detection of surface adsorption onto a piezoelectric crystal is based on Sauerbrey’s initial observation t h a t an oscillating piezoelectric crystal resonance frequency changes linearly with the mass of material which is surface 0003-2700/80/0352-1929$01 .OO/O
adsorbed (6). The frequency shift caused by adsorption is described by the equation where 1F is the change of frequency, F is the resonant frequency of the crystal, AM8is the change in mass of the surface, p is the density of the crystal material, A is the surface area being affected, and N is a constant. Adsorption measurements utilizing this technique are more difficult to obtain in a liquid than in a gas phase. Greater energy losses a t the liquid-crystal interface make crystal oscillation more difficult to maintain ( 7 ) . Oscillation frequency is also dependent on the density of the gas or liquid phase a t the crystal surface, and thus density changes from solvent gradients and solute cause a drift in resonant frequency. Finally, when working in the gas phase, liquid coatings may be employed to control crystal surface adsorption properties. Such coatings will wash off into a liquid phase, and alternate methods of controlling surface adsorption must be found. Despite these problems piezoelectric mass detectors offer some appealing advantages with respect to LC detection. Proper control of surface adsorption properties would allow the detection of general or specific classes of compounds. The knowledge and theory of how compounds are retained on LC columns may be applied to the problem of adsorbing compounds onto detector surfaces. Such an approach to detection is also potentially quite sensitive because small changes in crystal resonance frequency are easily detected. In the case of a GC piezoelectric detector, it was estimated that as little as lo-’ g of vapor-phase material could be detected ( 3 ) . In the preliminary study reported here, it was our goal to determine if the difficulties associated with t h e use of piezoelectric crystals in the liquid phase could be diminished or overcome. It was found that stable oscillation of quartz crystals in contact with a liquid phase can be achieved. Compensation for liquid density changes can also be made by employing reference and indicator crystals. T h e bonding of long chain hydrocarbons to the surfaces of quartz crystals was also found to be a potential approach to the control of surface adsorption properties in the presence of a liquid. Preliminary results with regard to the response of such a system to simulated LC conditions (me reported below.
EXPERIMENTAL SECTION Crystals and Cell. Piezoelectricquartz crystals were obtained from Piezo Crystal Co., Carlisle, PA. The crystals were 8.9 mm in diameter, 0.25 mm thick and had a resonance frequency of 7.00 MHz in air at 25 “C. After surface modification, Ag electrical contacts with a Ni overlay were deposited on the surface as round spots of approximately 5 mm in diameter. Such crystals were mounted inside of steel washers and placed on either end of a cylindrical detector cell. Au-plated springs were employed as external electrical contacts and as a means to hold the crystals in position as shown in the cell drawing of Figure 1. Solvent was forced to flow through the cell via the syringe needle connections shown in Figure 1 to simulate chromatographic conditions. Solvent was gravity fed from a buret. Solute was introduced by injecting small volumes 01’ dissolved solute into the C 1980 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 12, OCTOBER 1980
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Gerhart (8)was utilized to bond octadecyltrichlorosilane (Aldrich) to the surface to obtain surface sites of the type surface--Si-O-Si-C18H37 In the second approach docosyldimethyl(dimethy1amino)silane and trimethyl(dimethy1amino)silane(obtained from the laboratory of E. sz. Kovats) were surface bonded. In this method the crystals were first cleaned and activated by soaking in 65% "OB. The crystals were coated with the appropriate compound under Nz and held at 100 "C for 10 h under vacuum. It was expected that unmodified crystals or those treated with trimethyl(dimethy1amino)silane would exhibit little or very nonspecific surface adsorption. Crystals of this type were used as reference detectors to compensate for resonance frequency shifts caused only by liquid density changes. Crystals treated with the CI8 and CZ2hydrocarbons were employed as indicator detectors. More extensive and more specific surface adsorption was expected to occur with these crystals, causing frequency shifts in excess of that seen with the reference crystals.
FREQUENCY COUNTER
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Figure 2. Block diagram of experimental system.
solvent stream passing from the buret to the cell as is done in flow injection analysis. Electronics. Indicator and reference crystals were placed in separate crystal-controlled, FET transistor, Pierce oscillator circuits. Allowing only one face of each crystal to come into contact with the liquid phase was found t o permit stable oscillation. The signal from each crystal oscillator was frequency divided down through a series of digital binary counter circuits (74197's). The frequency of one crystal-controlled oscillator (indicator) was divided by a factor of 8, 16, 32, or 64, and the signal was then connected to the counter input of a digital electronic counter (Hewlett-Packard 523CR). The frequency of the second oscillator (reference) was divided by a factor of Zz2, 223,P4,or 225yielding 3 much lower frequency signal. This low-frequency signal was employed to control the gate of the digital counter. This arrangement of driving counter and gate with the two oscillator signals produces a periodic digital count which reflects the ratio of the two frequencies. The sensitivity of measurement of this frequency ratio may be varied by adjusting the division factors employed with each circuit. To obtain an analog signal proportional to the digital ratio for strip chart recording, a digital to analog convertor (HewlettPackard 580A) was interfaced to the counter. Figure 2 illustrates the overall arrangement of the experimental system. Surface Modification. Two different methods were employed to bond hydrocarbon molecules to the quartz surfaces of the crystals. In the first approach the method of Kingston and
RESULTS AND DISCUSSION Solvent Density Effects. The effect of changes in density of the liquid passing through the detector cell on the resonance frequency of a n oscillating piezoelectric quartz crystal was determined by placing only an untreated indicator crystal in the cell while keeping a reference crystal in air. Under this arrangement variations in the indicator/reference frequency ratio must be due to either density changes or nonspecific surface adsorption. Figure 3A illustrates the variation of be frequency ratio when 0.2 mL of 10% CCl, and 0.2 mL of 10% toluene (both dissolved in mobile phase) were injected into a mobile phase of 20% (v/v) aqueous methanol. Given a density of 0.97 g / m L for the mobile phase, the injection of more dense CCl, (1.59 g/mL) caused a decrease of 128 Hz in the resonance frequency. In contrast the introduction of less dense toluene (0.87 g/mL) caused a 40 Hz increase in frequency. These frequency shifts must be attributed primarily t o increases and decreases in liquid density a t t h e crystal surface. Any significant surface adsorption would cause a consistent decrease in crystal resonance frequency, in contrast to the behavior observed. When two nonadsorbing crystals were employed as indicator and reference crystals, the frequency of each crystal shifted proportionately upon a density change in the liquid. As a consequence the measured indicator/reference frequency ratio was found to remain relatively constant. For example, t h e injection of 0.2 mL of 10% naphthalene, which was not adsorbed by any crystal, did not cause a frequency ratio change outside normal base-line variations. Such base-line noise was found to be typically 3 ppm peak to peak, with respect to the unit ratio of the undivided frequencies during stable oscillation. Base-line fluctuations are graphically included in Figure 3. I t can be seen, therefore, that while liquid-phase density
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Anal. Chem. 1980, 52, 1931-1934
variations do effect the frequency of piezoelectric crystal vibration in the detector cell, such effects can be compensated simply by including a reference detector in the system. S u r f a c e Adsorption Effects. To detect surface adsorption, we placed indicator crystals bonded with either the CIS or Czzhydrocarbons in the detector cell as indicator crystals while plain as well as the trimethyl(dimethy1amino)silane bonded crystals were used as reference detectors. Figure 3B indicates the change in the indicator/reference frequency ratio after injection of 0.2 mL of 10% CC14 and 0.2 mL of 10% toluene into a mobile phase of 20% aqueous methanol. In the case of CC1, both crystals decreased in frequency due to a liquid density change. However, the CI8bonded indicator crystal exhibited a larger decrease because surface adsorption also occurred. This disproportionate change in the frequency of the indicator crystal caused the variation in the frequency ratio observed. T h e injection of toluene caused only the reference crystal to increase its frequency because of a decrease in liquid density. Surface adsorption a t the indicator crystal has predominated over density effects and lowered the indicator frequency causing a change in the frequency ratio. While detector response was observed for injections of C C 4 and toluene under these experimental conditions, the introduction of large nonpolar molecules such as oleic acid, butylbenzene, and naphthalene gave no adsorption response. The frequency shift of CI8 crystals was also found to be greater than for C Z 2bonded surfaces. Clearly what modification of the quartz surfaces t h a t was achieved allowed detectable adsorption of only small nonpolar molecules and with poor efficiency. Such a response does prove, however, that the effects of adsorption on crystal resonance frequency can be separated from those attributable to liquid density variations.
Such results also indicate that the selective detection of various solute classes is potentially possible. Further development of this approach toward LC detection must first center on improved methods of surface modification to encourage adsorption. It can be calculated from Sauerbrey's equations and constants (6) that the indicator crystal response to CCll shown in Figure 3B was caused by the absorption of only 1.3kg of the 31.8 mg of solute present. This low efficiency and sensitivity are also complicated by the fact that the treated crystals became saturated with solute after several consecutive injections and ceased giving a response. Soaking in a nonpolar solvent was found to regenerate the crystals, indicating that slow desorption rates are probably the cause of such behavior. The most likely origin of the above absorption problems is poor coverage of hydrocarbon molecules on the quartz surfaces. Improved surface modification techniques may therefore be expected to yield more sensitive detection, and further research efforts will be pointed in this direction. ACKNOWLEDGMENT We wish to thank E. sz. Kovats for the provision of two silane derivatives and for his helpful advice. LITERATURE CITED ( 1 ) Huber, J. F. K. "Instrumentation for High-Performance Liquid (2) (3) (4) (5) (6) (7) (8)
Chromatography"; Elsevier: Amsterdam, 1978. Kissinger, P. T. Anal. Chem 1977, 49. 447A King, W. H. Anal. Chem. 1964, 36, 1735. Karasek, F. W.; Gibbins, K. R. J . Chromatogr. Sci. 1971, 9 , 535. Hlavay, J.; Guilbault, G. G. Anal. Chem 1977, 49, 1890 Sauerbrey, G. Z . Phys. 1959, 155, 206. Webber, L. M.; Guilbault, G. G. Anal. Chim. Acta 1977, 193, 145. Kingston, P. I.; Gerhart, B. R. J. Chromatogr. 1976, 116, 182.
RECEIVED for review June 9, 1980. Accepted July 2, 1980.
High-Performance Liquid Chromatographic Determination of 5-Halopyrimidinone Interferon Inducers M. A. Wynalda and F. A. Fitzpatrick" Drug Metabolism Research, Unit 7256. Pharmaceutical Research and Development, The Upjohn Company, Kalamazoo, Michigan 4900 1
High-performance liquid chromatography with microparticulate, bonded, reversed-phase columns separates closely related 5-halopyrimidinones that are interferon inducers. A method was developed to quantitate serum levels of 2-amino-5bromo-6-phenyl-4(3H)-pyrimidinone, an important analogue of this new series. The method is, with minor modifications, suitable to measure other 5-halophenylpyrimidinone analogues. Results show that the quantitation of serum levels as low as 2 pg mL-' is possible with ultraviolet detection at 235 nm. Protein precipitation and extraction prior to chromatography improve the dally sample throughput by removing interfering peaks with capacity factors greater than 45. Preliminary results Indicate a species-dependent variation in the half-lives of elimination of the free compound after its administration, orally, to experimental animals. Rats clear the drug with a half-life of 4.5 h; cats clear the drug with half-lives ranging from 10 to 18 h depending on the dose administered. The differences in metabolic clearance may be relevant to observed toxicity dlfferences between these species. 0003-2700/80/0352-1931$01 .OO/O
Recent clinical trials indicated that interferon could be an effective antiviral and antineoplastic agent. Although this glycoprotein expresses its biological activity a t subnanogram levels, it is difficult to produce and purify. Despite its demonstrated potential, shortages of interferon hinder the expansion of clinical trials. Various conventional and unconventional mass production techniques could, conceivably, alleviate this shortage; however, they have not done so yet. Another approach to evaluate and develop the therapeutic potential of interferon relies on the inherent capacity of cells to secrete the substance, endogenously, after exposure to an inducing agent t h a t can derepress the, ordinarily, silent interferon gene. Whether one induces endogenous interferon production or whether one produces it for exogenous administration, the induction step and some form of interferon inducer are pivotal. Nevertheless, the development of safe and effective interferon inducers is a t a n embryonic stage. 5-Halopyrimidinones can induce interferon in vivo in several species, and in vitro in mammalian cell cultures, including 1980 American Chemical Society
