Anal. Chem. 1992, 64, 991-996
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Membrane Interfaces for Sample Introduction in Capillary Zone Electrophoresis Liyuan Bao and Purnendu K. Dasgupta* Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061
Small lengths of narrow-bore tubular membranes can be Interposed In the separation crrpilary in capllary electrophoreHc separatlon systcnns. These membrane can be used as "piingIntetfaceq a jacket k buHt outside the membrane, and the sample tS introduced by dWusion/permeatlon through the membrane. Various examples are shown; the determination of gaseous samples through a porous membrane, the determination of lonlzabie/nonlonic solutes by permeation through a diicone rubber membrane, and the separation of low MW constituents in blood plasma by transporl through a dlalyds membrane. I n the first two cases, significant preconcentration Is posdble, lhus permitting attractive detectlon limits.
High separation efficiencies, small sample needs, and simple equipment requirement have made capillary zone electrophoresis (CZE) of great current It is generally agreed that improvements in sample handling and introduction and reduction of limits of detection (LODs) in the conventional absorbance detection mode can benefit its present practice. Although laser-based detection of a fluorescent analyte can be exquisitely sensitive,6y7it is not always directly applicable. Although the sample requirement in CZE is small, sample availability is often not a problem; LODs could be improved if CZE was amenable to on-line preconcentration as in liquid chromatography. Under favorable conditions, electromigrative introduction of analyte ionse or operation under "stacking conditionsnQcan be used to improve LODs; these approaches are not likely to be universally applicable. With regard to sample pretreatment, CE has the usual requirements of a capillary format: the sample must be scrupulously filtered, etc. The major interest of our laboratory lies in the analysis of environmental samples; we can benefit from the separation efficiencies of CE, but the necessity for sample pretreatment poses a problem since the concentration of trace analyh are often a f f d during such manipulations. Membrane interfaces are capable of interphase transfer, matrix isolation, and preconcentration. They have been used with gas, liquid, and ion chromatography and flow injection a n a l y s i ~ The . ~ ~interface ~~ is typically part of an automated sample introduction system. Because of pressure limitations, membrane interfaces must be connected on the low-pressure side of auxiliary valves, e.g., on the loading conduit of a loop-type sample injector in a chromatograph. In a CZE system, the membrane interface can be in-line because high pressures are not involved. In this paper, we demonstrate membrane-interfaced CZE systems utilizing porous, permeative, and dialysis membranes and show applications ranging from the determination of atmospheric trace gases to the analysis of wastewater and constituents in blood plasma. EXPERIMENTAL SECTION Equipment. All electropherogramswere obtained on a CES-I system (Dionex Corp., Sunnyvale, CA) equipped with an oncolumn variable-wavelengthUV-vis detector. The instrument 0003-2700/92/0364-0991$03.00/0
was typically operated in the constant voltage mode (ca. 20 kV) with positive polarity. Fused silica capillaries (75-pmi.d. X 145or 375-pm 0.d.) were obtained from Polymicro Technologies (Phoenix, AZ). Auxiliary measurements were performed with a Dionex DX-100 ion chromatograph and a Hewlett-Packard8451A spectrophotometer. Reagents and Samples. Chemicalsused as analyte standards and buffer components were typically reagent grade or better. Solid NH4NOzwas prepared according to ref 10. For the analysis of trace gases, the electrophoretic buffer was 1.8 mM K2Cr207, 2 mM NazB407,40 mM H3B03, and 1mM diethylenetriamine (pH 7.8) with indirect detection at 274 nm. With HN03 (9) and HONO (g), direct detection at 204 and 212 nm, respectively, was possible, the same buffer as above was used except without K2Cr207.For SO2 (g), an 1.8 mM solution of K2Cr207was used in the indirect detection mode. Several different buffers were used for the analysis of aqueous samples. In the micellar electrokinetic capillary chromatography (MECC) mode, the buffer consisted of 50 mM sodium dodecylsulfate (Bio-Rad, Richmond, CA), 10 mM Na2B407,and 50 mM (pH 8.1). Borax (12.5 d, pH 10.8) and (NH4)$04 (5 mM, pH 3.2) were t y p i d y used as the electrophoretic media for the analysis of membranepermeable acidic and basic components, respectively. For the analysis of s m d molecular weight Components in blood plasma, the buffer used was the same borate buffer as above. For all the above examples, direct detection at 215 nm was used. All solutions were prepared in distilled deionized water of specific resistivity 1 18 m k m . Calibrant S02(g)was supplied by a permeation tube; calibrant sources of HNOZ(g)and HN03(g) have been previously described.lBHydrogen chloride, HCOOH, and CH3COOH gases were generated by passing Nz through a porous polypropylene tube (Accurel, Enka AG, Wuppertal, Germeny) immersed respectively in aqueous solutions of 2 M HC1, 0.005 M HCOOH, and 0.01 M CH3COOH. Similar calibrant generationsystems have been dea~ribed.~~-'~ All generationvessels were thermostated at 23.2 0.2 "C. Calibrant concentrationswere determinedby bubbler collection in water or 5 mM NaOH absorbers followed by ion chromatographicanalysis. Gravimetxic calibration of the permeation source was used for SOz. At a generation flow rate of 50 mL N2/min, the primary output concentrations (diluted as desired by additional N2 downstream) of HONO, HN03, SOz, HC1, CH3COOH, and HCOOH(g) were 530,880,560,3000,2040,and 760 parts per billion by volume (ppbv),respectively. Sampling was conducted at the desired flow rate, any excess source flow was vented. Membrane Interfaced Capillary Conduits. The membrane tube connected an 11-cm (to high voltage end) and a 45-55-cm segment (to detector and grounded end) of silica capillary tubing. The first segment should be ideally short and of larger bore to minimize voltage drop across it (sample is introduced after this); the dimensions chosen were largely dictated by the configuration of the particular commercial instrument used. Figure 1shows the manner in which the three different membranes were connected to the silica capillaries. For a porous polypropylene interface, each end of a 3-35" length of Celgard X-10 fiber (100-mi.d., 140-pm o.d., Hoechst-Celanese, Charlotte, NC) is inserted to a depth of 1mm inside each of two 250-pm4.d. poly(ethy1vinylacetate) (PEVA, micro-line tubing, Cole-Parmer, Inc., Chicago, IL)tube segments,ca.4-5 mm long. A hot soldering iron tip is brought close to the PEVA tube housing the membrane; the PEVA tube softens and shrinks, forming a seal. The silica capillary is forcibly inserted in to the free ends of each PEVA tube to butt against the inserted membrane tube at each end. 0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 9, MAY 1, 1992
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Flgure 1. Connecting different membranes to silica capillaries. (a) Porous membrane interface. A, A': silica capillaries, 75-pm i.d., 375-pm 0.d. P: PEVA tube segment, 250 pm i.d. M: 100-pm i.d., 140-pm 0.d. porous polypropylene membrane, exposed length 1-1.5 mm. (b) Silicone membrane interface. A, A': silica capillaries, 75pm i.d., 375-pm o.d., forced into M (originally 250-pm i.d., 450-pm 0.d.) silicone rubber tube. A and A' are separated by 0.1-0.2 mm. (c) Dialysis membrane interface. A, A': silica capillaries, 75-pm i.d., 145-pm 0.d. M: 200-pm4.d. cellulose acetate dialysis fiber, A and A' are separated by 1 mm. E: epoxy adhesive. (d) The sampling interface with jacket installed. A, A': silica capillary, source and detector ends, respectively. B: sealant, typically poly(viny1chloride) tube segment. C: tee. D: PTFE jacket tube.
Epoxy adhesive applied around the PEVA tube-membrane junction prevents leaks. The active length of the membrane is 1-1.5 mm. For a silicone rubber interface, a 5-mm tube segment (250-pm i.d., 450-pm o.d., Patter Products, Beaverton, MI) is swollen in hexane and the capillary termini (75 X 375 pm) are inserted to form a virtual butt joint. The induced stretching reduces the membrane thickness from the original value of 100 to -80 pm. Microscopic examinationshows that an 100-200-pm gap is typically left between the capillary termini because of the bowing of the membrane. A cellulose acetate dialysis interface (Spectrapor HF hollow fiber, 180-pm id., 200-pm o.d., molecular weight cutoff 5000, Spectrum Medical Industries, Los Angeles, CA) requires care because of the fragility the fiber. Silica capillaries (75-pm i.d., 145-pm o.d.), held by independent positioners, are inserted at each end of the membrane segment, and epoxy adhesive affiies the membrane to the capillary. During installation of the jacket (vide infra), a few centimeters of a second capillary should be used temporarily as a support around the membrane. To complete device construction, it is necessary to install a jacket around the membrane to allow the passage of sample. Figure Id shows the arrangement, based on subminiature polypropylene tees (Ark-Plas Inc., Flippin, AR). Sampling Arrangement. For the porous membrane system (Figure 2a), gas samples were allowed to flow through valve V and the sampling jacket, the flow rate was controlled by needle valve N in the vent line and measured by the digital soap bubble meter DSM. Switching valve V (3-way all PTFE solenoid valve, 075T3WMP 12-32,Biochem Valve Corp., Hanover, NJ) allows a dry zero gas D to flow in the jacket. For the silicone membrane system (Figure2b),the sample S is aspirated by a peristaltic pump P (Minipuls 2, Gilson Medical Electronics, Middleton, WI) through J and switching V causes the stream to switch to a wash solution W. For some experiments, a second valve was added in-line to the first one to aspirate air after the wash step and minimize sample carryover. For the dialysis membrane interface (Figure 2c), the sample was aspirated through the jacket followed by a wash with water or the electrophoretic buffer. Air was then admitted through the second valve V2 to remove all liquid from the jacket. When electrophoresis begins, air is stationary in the jacket.
Flgure 2. Sampling arrangement used with (a) porous, (b) silicone, and (c) dialysis membranes. GS: Gas sample source. N: Needle valve. V, V1, 2: 3-way PTFE solenoid valves. J: Capillary jacket. DSM: Digital soap bubble meter. S: Liquid sample. W: Wash solution. P: Peristaltic pump.
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5min Flgure 3. Analyte signals from the sampling of trace gases through porous membranes: all flow rates are 100 mL/min and the sampling periods are 2 min: (a) 440 ppbv HNO, direct detection; (b) 3000 ppbv HCI, indirect detection; (c) 380 ,ppbv HCOOH, indirect detection; and (d) replicate signals from 265 ppbv HONO, direct detection. Vertical bars indicate 0.0005 absorbance unit in all cases.
RESULTS AND DISCUSSION Measurement of Gases with a Porous Membrane. Typically, following sampling for the desired period, zero gas (dry air, N2or He) flow is used to sweep off remaining sample gas, and electrophoresis is begun. Typical electropherograms showing the analyte signals are shown in Figure 3; the detection mode (direct vs indirect) would appear to be the major determining factor in governing S / N and hence attainable LODs. Mixtures of gases could be analyzed, e.g., the signals from HN03 and HONO, and HC1 and HONO were resolved, using direct and indirect detection modes, respectively. Distinct tailing, not related to a specific gas, is also apparent in some cases in Figure 3. During gas sampling with a stationary receptor liquid, microbubbles are sometimes formed at the inner surface of the membrane. The resulting inefficient washout pattern is likely responsible for the tailing.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 9, MAY 1, 1992
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25.0 ~ H N O J 440 , ppbv m H O N O . 265 ppbv M & U H C I . 3000 ppbv fif&i!&HCOOH, 380 ppbv
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Sample Flow, mL/min Flgure 4. Analyte response as a function of gas sampling rate at constant concentratkn,porous membrane interface, 2 mln per sample.
Flgrvo 5. Response as a function of preconcentratkm time for varlow test gases, porous membrane interface.
Note that water vapor efflux through the porous membrane, aided by Joule heating of the liquid, can lead to condensation in the jacket during electrophoresis. Unless this is removed before subsequent sampling, sample gases are removed by this liquid. Dry zero gas flow, either between or continuously during electrophoretic runs, is used to remove or prevent condensation. A continuous flow of 30 mL/min is sufficient for this purpose. Collection Efficiency as a Function of Sampling Rate. For an annular device where the membrane tube is the central element and gas flow occurs outside it, collection efficiency decreases with increasing flow rate in a manner that the signal response (proportional to the product of the collection efficiency and the flow rate) becomes essentially constant past a certain flow rate.20 Such a "plateau response" is attained at quite low flow rates in the present case, as the data in Figure 4 show for HC1 and HONO. This behavior is convenient because precise flow control becomes unnecessary. The reaults for HN03 are different. We believe that this is due to the extremely tenacious adsorption behavior of this analyte.16 Nitric acid may simply be transmitted through the connecting conduits up to the membrane more efficiently at higher flow rates. Response Linearity. Response to HN03, HONO, HC1, and HCOOH was tested at concentrations of 0-440,0-265, 0-1625, and 0-380 ppbv respectively, all at a flow rate of 100 mL/min. A linear response was observed in all four cases, with near-zero intercepts and good linear correlation coefficients (0.99524.9982). Response as a Function of Sampling Time. The increase in response as a function of sampling time was determined for several gases and is shown in Figure 5. A linear relationship is expected and is observed for HC1 and HCOOH. The nonlinear behavior exhibited by HN03 and HONO in the present system is not understood; linear response behavior with increasing preconcentration times has been observed previously for HN03 and HONO collection experimenta with similar porous membrane based devices built on a larger scale and coupled to chromatographs.10 Reproducibility and Limits of Detection. The reproducibility of individual measurements was ascertained by repeated measurements of HN03, HONO, HC1, and HCOOH at the same test concentrations and flow rates as in Figure 5 using a sampling time of 2 min. The relative standard deviations (RSD) were 2.4, 4.2, 13.5, and 6.6% for the respective gases. Note that this uncertainty includes any instabilities of the generation source. The RSD of the HONO
measurements is in keeping with the previously observed intrinsic stability of this source (RSD 4.6'70, ref 10). No information is available on the stabilities of the type of HC1 and HCOOH sources used here. The high RSDs observed in the HC1 experiments is most likely due to the difficulties of preventing contamination from trace levels of chloride. For comparison, gravity injection (100 mm, 30 s) of the corresponding sodium salts at the 5-10 mg/L level produced RSD values of 3.7, 2.3, 3.9, and 3.2% for NO,, NO2-, Cl-, and HCOO-, respectively. Based on the results with HN03 and HONO, intrinsic reproducibility problems are not indicated. The limit of detection is obviously dependent on the sampling period. At a flow rate of 100 mL/min and with the concentrations denoted in Figure 5, a 2-min preconcentration period showed S/N levels that lead to respective LODs (S/N = 3) of 9.6,10.3,21, 51,81, and 440 ppbv for HONO, HN03, S02, HC1, HCOOH, and CH3COOH. Measurement of Ionizable/Nonionic Compounds with a Silicone Rubber Membrane. Preconcentration of an ionizable protic compound in a sample is poasible by adjusting the pH to convert it into the unionized state in which form it is permeable through a silicone membrane,13-15,21-22The receptor pH is chosen to promote analyte ionization; since the charged form does not partition to the membrane, an one-way transfer path is established. Obviously this principle cannot be applied to a nonionic analyte. However, a mechanism for preconcentration is established if a micellar solution is used as the receptor because the partition constant for a typical neutral organic molecule from a purely aqueous sample into a micellar pseudophase can be quite large. The intial use of the silicone interface showed, however, a considerable amount of peak tailing and a major rise in the baseline. The first responsible factor is the space between the walls of the two silica capillaries that acts as a reservoir. Analytes permeate into this (stagnant?) zone and must then diffuse into the principal flow stream during electrophoresis. Second, transmembrane transport is slow. Even after sampling is ceased,analytes continue to diffuse from the reservoirs represented by the dead zone and the membrane; this leads to tailing. We reasoned that washing the jacket volume with a "desorbing" solution following the sampling period may be of value. The effectiveness of an aqueous acetonitrile solution (80:20 H20/CH3CN) in reducing the magnitude of this problem is illustrated in Figure 6 for a sample containing several phenols (acidified sample, alkaline receptor). Although a large amount of CH3CN is introduced into the capillary,the peak is relatively small and is generally well separated from
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 9, MAY 1, 1992 CEIZOo-Cresoi, pK 10.26 m-Cresol, pK 10.09 M P h e n o l , pK 9.99 2,4-Dichlorophenol nK 7.8s =h-N;
