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AMI. Chem. 1992, 64, 2461-2464
TECHNICAL NOTES
Cellulose Acetate-Coated Porous Polymer Joint for Capillary Zone Electrophoresis Chen-Wen Whang’ and I-Chih Chen Department of Chemistry, Tunghai University, Taichung, Taiwan 40704, Republic of China
INTRODUCTION Capillary zone electrophoresis (CZE) has become an important separation method as a result of its high resolving power and speed. Separations with more than 106theoretical plates in less than 20 min has been demonstrated.l.2 Due to the extremely small volumes of sample zone encountered in CZE, on-column detection, such as UV absorption and fluorescencedetection, are the most commonlyused methods. On-column detection eliminates zone dispersion owing to the joints, fittings, and connectors that are necessary for conventional off-column detection systems. However, in some applications, such as CZE coupled with off-column electrochemicaldetection or continuoussample collection,acapillary outlet free from the applied potential field is generally required. To isolate the column end from the high voltage, special deviceslike a porous glassjoint? porous graphite joint,’ and on-columnglassfrits have been applied to create a currentfree zone at the capillary outlet, where an electrochemical sensor or a fraction collector is placed. Although these techniques work well with direct electrochemical detection and off-column sample collection, the problem is how to produce such structures reliably and inexpensively. Recently, Linhares and Kiasinge$ reported a sample introduction system for CZE based on a bare on-column fracture near the inlet end of the capillary. When a potential is applied between the fracture and the outlet end of the capillary, electroosmotic flow pulls sample into the capillary through the inlet. This sample introduction system can be viewed as an ‘electroosmotic syringe”. The on-column fracture is easy to construct, and it can also be used as a general method to separate the column into two segments. We have tried using this technique to create a current-free segment of capillaryfor off-columnelectrochemicaldetection. However, we did observe some sample leakage through the fracture assembly. This is probably caused by the backpressure generated inside the shorter segment of capillary because of the laminar type of flow in it, which forces the analyte ions to diffuse through the open fracture. In this paper, we describe a modified capillary joint for CZE. Inatead of usinga bare fracture as the coupler, a cellulose acetate (CA) membrane was uniformly coated over the fracture. This design is basically similar to the porous glass joint used by Ewing,3 except that a porous polymer joint is created here. CA membranes are known to be permselective primarily based on size; only small molecules (H202,02) and ions (Na+, C1-, etc.) can diffuse rapidly through the mem-
brane.798 Larger analyte and solvent molecules are excluded from permeating through the pores. CA-coated electrodes have been commoly used by electrochemists to build a sizeexclusion selectivity into electrochemical detection in static solution9 and a flowing stream.10-12 Problems of electrode poisoning, due to protein adsorption or accumulation of reaction products, can be effectively eliminated. In the present case, the CA-coated fracture will ensure that only small buffer ions and, therefore, current will pass but does not allow the larger analyte ions to pass through it. In comparison with a bare on-column fracture, the CA-coated porous polymer joint provides better efficiency and minimal sample loss. Besides, it has the advantages of long durability, inexpensivenew, and easy construction.
EXPERIMENTAL SECTION Apparatus. The CZE system was assembled in-house. A high-voltage power supply (Glassman High Voltage, Inc., Whitehouse Station, NJ;Model PWEH40R02.5) was used to generate the potential across the capillary. Fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) of 50-pm i.d., 360-pm o.d., and 70-cm length were used in this study. After the polymercoatedjoint was formed (described below),the capillary was fiied with buffer solution. The high-voltage end of the capillary and the buffer reservoir were contained in a Plexiglass cabinet equipped with an interlock for operator safety. Platinum wire was used as the electrode. Detection of UV absorption was performed at 254 nm using an UV detector (LinearInstruments, Reno, NV; Model UVIS 200) equipped with an on-column capillary cell module. The output signal was recordedwitheither a strip-chart recorder (Pan&, Kyoto, Japan; Model U-228) or an integrator (Shimatzu, Kyoto, Japan; Model C-RIA). In the determination of possible leakagefrom the porous polymer joint, a constant-flowHPLC pump (Eldex,San Carlos, CA; Model 9600) and a fluorescence spectrophotometer (Hitachi, Tokyo, Japan; Model 160-105)were used. Reagents. All chemicals were of analytical-reagent grade. Thiamine,nicotinamide, and riboflavin were obtained from Sigma Chemical Co. (St. Louis, MO). Pyridoxal hydrochloride and sodium fluorescein were purchased from E. Merck (Darmstadt, FRG). Cellulose acetate (39.8% acetyl content) was obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI). A 12% (w/v) cellulose acetate (CA) solution was prepared with HPLC-grade acetone. The electrophoresis buffer was a mixture of 10 mM sodium dihydrogen phosphate and 10 mM sodium tetraborate, pH 9.0. The buffer solution wasprepared with dietilled4eionized water, obtained using a Sybron/Barnstead (Boston, MA) NAN(7) Lonsdale, H. K.; Croes, B. P.; Graber, F. M.; Milstead, C. E.In Permelectwe Membranes;Rogers,C.E., Ed.;MarcelDekker: New York,
_.
1971: DD 167-187. (8) Colton, C. K.; Smith, K. A.; Merrill, E. W.; Farrel, P.C. J.B i o m d . Mater. Res. 1971,5,459-486. (9) Kuhn, L. S.; Weber, S. G.; Iamail, K. Z. A d . Chem. 1989, 61, 303-309. (10) Sittampalam,G.; Wileon, G. S. Anal. Chem. 1983,55,1608-1610. (11) Wang, J.; Hutchine, L. D. Anal. Chem. 1985,57,1536-1541. (12) Hutchine, L. D.; Wang, J.; Tuzhi, P. Anal. Chem. 1986,58,101* 1023. Cr
(1) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981,53,1298-1302.
(2) Jorgenson, J. W.; Lukaca, K. D. Science 1988,222,266-272. (3) Wallingford, R.A.; Ewing, A. G. AM^. Chem. 1987,59,1762-1766. (4) Yik,Y.F.;Lee, H.K.;Li,S.F. Y.;Khw,S.B. J.Chromatogr. 1991, 585,13%144. (5) Huang, X.;Zare, R. N.J. Chromatogr. 1990,516, 185-189. (6) Linhares, M. C.; Kiesinger, P. T. Anal. Chem. 1991,63,2076-2078. 0003-2700/92/0364-2461S03.00/0
@ I9Q2 Amerlcen Chemlunl Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 20,
OCTOBER 15, 1992
o.oozA
a-
2
b-+ 1
C -
d
1
0 0 0
a
Flgure 1. Cellulose acetatacoated fracture assembly and buffer reservoir: (a) fused-slllca caplliary; (b) piatlnumwhe electrode;(c) 10-mL plastic vial; (d) buffer solutlon; (e)epoxy glue; (f) CAcoated fracture; (9) glass plate.
Opure I1 system. The solution was filtered through a 0.45-pm membrane filter prior to use. Cellulose Acetate-Coated Capillary Joint. The procedure for construction of an on-column fracture was similar to that described by Linhares and Kissingera6A small section (-3 mm) of polyimide coating was burned off 9 cm from the end of a 70cm-long fused-silica capillary. The capillary was placed over a 2-cm x 1-cmmicroscopeslide,on whichaV-shaped breach (about 4 mm wide and 3 mm deep) was created near the center of one edge. The exposed section of capillary was situated on top of the breach and was glued in place with epoxy glue (Spar Chemical Industry Co., San Francisco, CA) at each end of the exposed region. By using a glass-fiber cleaver (Newport;Fountain Valley, CA), a small scratch was made on top of the uncoated silica. The capillary was then pushed up gently from the bottom, directly under the scratch, with a small pointed stylet thereby forming the fracture. By use of a micropipet tip, a small drop (-3 pL) of 12% (w/v)CA solution (in acetone) was carefullydripped onto the fracture. Under a gentle stream of air and with slight rotation of the whole microscope slide, a thin film of CA membrane was uniformly coated over the fracture region. After drying.in air for 1h, the film thickness was found to be about 80 pm, as estimated under a microscope. The fracture assembly was then placed in a 10-mLplastic vial with a 1-mm hole drilled in the bottom. The microscope slide was vertically glued to the inner wall of the vial with the short section of capillary stretching out of the hole in the bottom. The length of the stretched capillary was about 8 cm. With the capillary in position, the hole was sealed using epoxy glue. The vial was then filled with buffer solution and a platinum-wire electrode was dipped in the solution. This electrode was connected in series to a digital current monitor (Keithley Instruments, Inc., Cleveland, OH; Model 177 DMM) and the ground terminal on the high-voltage power supply. A detailed schematic of the polymer-coatedcapillaryjoint is shown in Figure 1.
CZE Procedure. A detection window 4 cm either before or after the capillaryjoint was formed by burning off a small region of polyimide coating. The capillary was then carefully inserted into the UV detector, and the detection window was aligned with the focusing lens. The capillary was fist washed (pressurized flow) with 0.1 M NaOH for 3 min, followed by a 2-min rinse with water and a 2-min flush with the running buffer. Finally, the column was equilibrated with the buffer under and electric field
4 ~ 1 1 0 TIME (min) Flgurr 2. Electropherogram of four vitamins with UV detectbn 4 cm before the fracture. Conditions: column 50-pm 1.d. X 360-pm 0.d. X 7oCmtotal length, owcolumn fracture Q cm from the end of capulary; buffer solution 10 mM phosphete 10 mM borate, pH 9.0; vottage applied 23 kV (18 PA) across the anodk end of capllhry and the^ fracture;5-s gravlty injectbnat 1&cm height; W detectkm wavelength 254 nm. Peak identiths; (1) thiamine; (2) nicotlnnmkk; (3)rlbofhvhr; 0
2
+
(4) Pyridoxal.
of 200 VJcmfor 1h. Sample injection was performed by gravity. The capillary inlet was lifted 15 cm higher than the capillary outlet for 5 a.
RESULTS AND DISCUSSION On a fused-silica capillary with a thick wall (146 pm in this study), an on-column fracture assembly can be easily constructed following the published procedure.6 This fracture functions as a joint connecting two segments of capillary, which is generally required for sample collection, electrochemical detection, or other off-column detection methods in CZE. The fracture created is very fine, and the two sections of capillary are never separated. Since the fracture is so small, minimum bulk flow through the fracture should be expected. However, during a series of CZE experiments using capillaries with the fracture assembly for off-column electrochemical detection, we did observe some leakage of sample components through the fracture. In order to examine the characteristic of a capillary with a bare on-column fracture, two detection windows were created on the capillary with one at 4 cm before the fracture and the other 4 cm after the fracture. Four water-aoluble vitamins, viz.,thiamine (vitaminBI),nicotinamide,riboflavin (vitamin Bz),and pyridoxal (vitaminBe),were used astest compounds. In a buffer of pH 9.0, nicotinamide behaves as a neutral marker. High voltage was applied between the anodic end of capillary (in a Plexiglas box) and the cathodic end at the fracture assembly. A typical electropherogram of the four vitamins is illustrated in Figure 2. A significant decrease in peak size of the four compounds was observed when the electropherogram obtained after the fracture was compared with that obtained before the fracture. The losses of peak area range from 22 % for riboflavin (molecular weight = 376.4) to 38% for nicotinamide (molecular weight = 122.1). The average loss of N (theoretical plate number) is about 20%.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 20, OCTOBER 15, l9Q2 2468
Table I. Migration Times ( tm),Theoretical Plate Numbers (N), and Peak Area (A) of Four Vitamins Detected before and after the CA-Coated FractureP before the fracture compd thiamine nicotinamide riboflavin pyridoxal
tm, 8
222 (2) 288 (3) 330 (4) 426 (4)
after the fracture
A
104N 1.08 (0.18) 1.71(0.14) 1.60(0.21) 2.04 (0.24)
662 (34) 1271 (100) 1376 (159) 2635 (50)
tm, 8
104N
A
300 (2) 372 (3) 426 (5) 546 (3)
1.00 (0.23) 1.56 (0.22) 1.53 (0.15) 1.76 (0.12)
590 (31) 1232 (48) 1316 (102) 2582 (129)
Data are represented as mean (standard deviation); n = 5. Experimental conditions as in Figure 2.
Altogether we have tested four capillaries with a carefully constructed on-column fracture; none of them gave a loss of peak area smaller than 18% for nicotinamide, the compound with the smallest molecular weight (and size) in the test sample. We believe this is caused by leakage of sample through the open fracture, with components of smaller size leaking the most. It has been shown that the length of the secondcapillary behind the joint will have a pronounced effect on the column efficiency.3 This is because the volume of buffer within the second capillary must be driven by the electroosmoticflow generated in the separation capillary. The formed laminar flow of buffer within the second capillary will create a back-pressure which is proportional to the length of the second capillary. The optimal efficiency can be obtained with a short (
