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Anal. Chem. 1904, 56,2612-2613
Tubular Debubbler for Segmented Continuous-Flow Automated Analyzers Glenn B. Martin, Hee Kyoung Cho, and M. E. Meyerhoff* Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109 Since the invention of air-segmented continuous-flow analysis (CFA) by Skeggs (1)in the late 19509, the use of this technique for automated determinations of a wide range of species has fluorished. Indeed, until recently, automated analyzers such as the Technicon series have been the workhorses of most clinical and environmental laboratories. While newer flow-injection analysis methods are becoming more popular (2, 3), the CFA approach is still widely utilized, particularly for methods which require a relatively long incubation time to generate a detectable product from the analyte and appropriate analytical reagents (e.g., enzyme reactions, etc.). In CFA, the presence of air bubbles in the flowing sample-reagent stream serves to minimize dispersion by reducing longitudinal diffiision within the sample segment of the stream. However, prior to detection, the sample-reagent stream is usually “debubbled” in order to eliminate noise in the detector (i.e., spectrophotometric, electrochemical, etc.). Typically, the debubbling process occurs upstream from the detector via the use of a debubbler assembly such as that shown in Figure 1. Usually, the flow of the sample through the detector is controlled by a pull-through tube incorporated into the multichannel peristaltic pump. Because of the geometry of the debubbler apparatus, and the relative densities of the two phases, the air is prevented from passing through the detector and exits the debubbler to waste. Alternatively, a pullback on the air outlet stream can be utilized to accomplish the same result. Each of the above approaches requires the use of an additional channel within the peristaltic pump of the system-a channel which could better be used to deliver another reagent if needed. In addition, in order to avoid air bubbles in the detector originating from unwanted air unavoidably sampled by the automatic sampler arm as it transfers from the sample to wash solution, the flow rate of the pull-through tube is usually made less than the total flow rate of the liquid (sample and reagents) that enters the debubbler. Thus, part of the sample stream goes directly to waste (along with the air bubbles), having never passed through the detector. For detectors with slower response times, this reduction in sample size can prevent attainment of true equilibrium detector response and consequently reduce sensitivity (equilibrium response refers to the final detector signal generated if the sample was placed in the detector cell without use of continuous flow). We now propose a new debubbler which is rather simple in design yet provides a completely effective means of continuously debubbling air-segmented flowing streams. The proposed debubbler consists of a small piece of tubular microporous poly(tetrafluoroethy1ene) (Teflon) placed in the sample stream immediately before the flow-through detector. Pressure on the air bubble as a result of the pulsating peristaltic pump forces the air bubble through the tube, leaving the liquid to enter the detector. No additional pull-through tubes are required, and all of the sample-reagent liquid passes through the detector. Results obtained for an automated analyzer arrangement incorporating a flow-through ammonium ion-selective electrode detector verify the performance of the proposed device.
EXPERIMENTAL SECTION Apparatus. A schematic diagram of one of the automated analyzer manifold arrangements used to test the new debubbler device is shown in Figure 2a. The sampler and pump were ~
reconditioned Technicon components obtained from Alpkem Inc. (Clackamus, OR). The tubular ammonium-selective polymer membrane electrode, incorporating the antibiotic nonactin, was prepared as previously described ( 4 ) . Electrode potentials were measured with a Fisher Accumet Model 620 pH/millivolt meter and were recorded on a Houston Instruments Omniscribe stripchart recorder. All measurements were made vs. a saturated calomel reference electrode at room temperature. Samples were introduced into the system at a rate of 40/h with a 1:2 sampleto-wash ratio. The debubbler was constructed from a small piece of microporous poly(tetrafluoroethy1ene) tubing, approximately 1mm i.d., obtained from W. L. Gore and Assoc. (Elkton,MD). Two different pore sizes were examined; 3.5 and 2.0 pm. Typically, a 2-5-cm length piece of the tubing was incorporated into the flowing stream by inserting two small pieces of poly(ethy1ene) tubing into each end of the Teflon tube. The elastic nature of the Teflon formed a tight seal around the two end piece connectors. One end of the debubbler assembly was connected to the sample stream by inserting the poly(ethy1ene) connector into the end of the PVC transmission tubing used t o deliver the sample stream to the detector and the other to the inlet of the flow-through ammonium electrode detector. Reagents. Standards of ammonium chloride in the range of to 5 X 5 X mol/L were prepared in 0.01 mol/L tris(hydroxymethyl)aminomethane/sulfuric acid buffer, pH 7.5. These samples were placed in the sampler tray of the automated system shown in Figure 2a to evaluate the debubbler design.
RESULTS AND DISCUSSION A close-up view of the proposed tubular debubbler assembly is shown in Figure 2b. The hydrophobic nature of the poly(tetrafluoroethylene) tubing prevents effusion of the liquid sample through the pores. However, provided that there is ample back pressure on the air bubble, caused by the use of a flow restricter, or by simply elevating the outlet tube, the pressure placed on the bubble is greater than atmospheric pressure and this difference forces the air bubble through the porous tubing. We have found that it is best to place the exit stream (after the detector) above the height of the debubbler tube to provide the back pressure and to prevent siphoning of the liquid sample and air through the detector. This arrangement provides sufficient back pressure to completely debubble the stream. As expected, for a given length of tubing, we found that the more porous debubbler (3.5 pm) removed the air bubbles more effectively than the less porous Teflon. In addition, the length of the debubbler tubing required to completely remove the air from a segmented stream was dependent on the flow rate and volume of air in the stream. In general, we observed that no matter what the flow rates or size of the air segment was, a suitable length of tubing could be found that completely removed the air. Typically, a 5-cm length was more than adequate for most manifold arrangements. For example, Figure 3 shows a strip-chart recording of the potentials for the ammonium ion-selective electrode used as a flow-through detector in the automated analyzer system shown in Figure 1. Here, air was introduced at the rate of 2.0 mL/min to a sample stream flowing at 2.90 mL/min, and a 5-cm length of the porous Teflon tubing was used as the debubbler. Electrochemical detectors are extremely sensitive to even the smallest amount of air, and if any air had entered the detector, large potential spikes would have resulted in the output (i.e., open-circuit situation). As can be seen, there are no such spikes. The slight noise observed is due to environmental noise (from static electricity) which is typically encountered with
0003-2700/84/0356-2612$01.50/00 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
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---------------Flgure 2. (a) Manifold arrangement for ammonium electrode based automated analyzer system used to evaluate new debubbler: (ne) flow-through electrode, (sb) salt bridge, (mv) pH/millivolt meter, (rec) recorder, (r) saturated calomel reference electrode, (e) electrolyte solution, (9) electrical ground, (d) tubular debubbler, (w) waste. (b) Expanded view of tubular debubbler portion of manlfold.
the use of such detectors. A plot of peak potentials vs. the logarithm of the ammonium chloride concentration yields an electrode slope of 58.7 mV/decade (correlation coefficient, 0.9999) indicating that the electrode was achieving a nearly equilibrium Nernstian response in the flowing system. There are a number of advantages offered by the proposed debubbler. Firstly, there is no need for a pull-through tube in the peristaltic pump to control the flow of liquid through the detector. This leaves an additional channel of the peristaltic pump available to deliver other reagents. Secondly,
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Figure 3. Typical strip-chart recording for the automated analyzer system shown In Figure 2 as various solutions of ammonium chloride are sampled. Concentrations in micromoles per liter.
all the liquid sample goes through the detector before going to waste. This is desirable in instances where the detector response time is sluggish. Thus, by increasing the size of the sample slug that goes through the detector, improvements in sensitivity may result. Finally, the tubular debubbler can conveniently be placed very close to the inlet of the detector (within an inch or less), and this ensures that there is very little time for increased sample dispersion to occur after the segmentation has been removed. Since our earliest experiments (Figure 3), we have further examined the feasibility of using this debubbler design in a variety of other automated analyzer arrangements, employing either electrochemical or spectrophotometric detectors. In all cases, the debubbler design functioned as required and completely eliminated the air from the segmented streams. In view of the simplicity of the device and the advantages offered, we recommend that this new debubbler be considered by others who work regularly with segmented flow automated analyzer systems.
LITERATURE CITED (1) Skeggs, L. T. Am. J . Clin. Pathol. 1957, 28, 311-322. (2) Betterldge, D. Anal. Chem. 1978, 5 0 , 832A-846A. (3) Stewart, K. K. Anal. Chem. 1983, 55, 931A-940A. (4) Fraticelli, Y. M.; Meyerhoff, M. E. Anal. Chem. 1981, 5 3 , 992-997.
RECEIVED for review May 21,1984. Accepted July 9, 1984. This work was supported by the National Institutes of Health (Grant R01-GM-2882-04).
