In the Laboratory edited by
Topics in Chemical Instrumentation
David Treichel Nebraska Wesleyan University Lincoln, NE 68504
Microscale Capillary Electrophoresis: W A Complete Instrumentation Experiment for Chemistry Students at the Undergraduate Junior or Senior Level Ruben T. Almaraz* Biomedical Engineering Program, California State University, Sacramento, CA 95819-6039; *
[email protected] Maria Kochis CSUS Library Reference Department, California State University, Sacramento, CA 95819-6039
Teaching instrumentation to undergraduate chemistry students in the 21st century is a challenge as well as a necessity. For a variety of reasons, professors have to be selective in deciding which experiments they want to integrate into their classes and laboratory sessions. Experiments are primarily evaluated by the length of time to complete, the cost in terms of materials and instruments, and, most importantly, the inclusion of main, up-to-date techniques of chemical instrumentation. Undoubtedly, at one point or another in their career, future chemists will be required to set up, repair, or even construct a new analytical instrument. However, the anxieties experienced by students when venturing inside an instrument on their own, and the unlikelihood that they may have access to such devices outside the classroom, suggest that they must rely mainly on laboratory sessions to get acquainted with new analytical systems. For undergraduates, a typical chemistry laboratory session in instrumentation may include: constructing a circuit, troubleshooting an instrument, or changing the separation conditions of a sample and its matrix in order to optimize separation. Nowadays, most of these actions can be accomplished by interfacing with a computer screen. However, this does not provide the opportunity for students to explore the inner workings of an instrument which will greatly help them understand the instrument’s functionality. It is imperative that all students understand the most common and contemporary analytical and spectroscopic instruments, so that they can later be productive as professionals. A contemporary technique that has gained widespread acceptance in analytical laboratories is capillary electrophoresis (CE). CE is widely used by scientists for qualitative and quantitative analysis since it provides many advantages over conventional techniques such as NMR and HPLC. CE provides higher efficiency, lower sample consumption, and less waste production than conventional techniques (1). The experiment we present is relatively inexpensive because most of the components used in the construction of the instrument are conventional devices. It is also time efficient. The simplicity of the different tasks in the construction of the instrument allows every student in the class to fully participate in the experiment, which should not take more than three laboratory sessions to complete. Manz and Harrison are the pioneers of capillary electrophoresis on a microchip, and since their first publication 316
in 1992 (2), a multitude of new articles concerning the application of this technology have been published (3). Downsizing chemical analysis systems on planar substrates utilizing electrokinetic phenomena for sample separation has the same advantages as the conventional analytical systems, and in some cases is better in terms of the resolution and analysis time (3). As the chemistry students of today become professionals in the field, they will have increasing contact with this progressive technique. This is another incentive for incorporating it into university curricula. The experiment itself, in which the students are required to build their own capillary electrophoresis system on a microscope glass slide, is beneficial because it allows them to apply knowledge from various related areas of chemical instrumentation. During this experiment, students are required to construct a sensor consisting of a high-intensity LED as a light source and a photodiode, with an amplifier integrated in the same chip, as a detector. These experiments provide students with a basic understanding of both electronics and photo detection. In addition, students will gain experience working with glass by mechanically drilling microchannels in a microscope slide and thermally annealing and laminating a cover slide. These activities will familiarize students with some of the properties of glass and its use for analyte separation. Finally, students are exposed to data acquisition, a process by which they learn digital electronics with LabVIEW programming, LabVIEW waveform input, and signal processing. A simplified diagram set up of the system is presented in Figure 1. CE Construction
Materials Needed Linrose red super bright LED or any other high intensity red LED Two photodiodes (Opt 301, Burr Brown Corp.) Optical fibers (20 cm) Platinum wires (30 cm) 714 operation amplifier and resistors Four banana-plug connectors Wooden box with a minimum interior volume of 150 cm3
Journal of Chemical Education • Vol. 80 No. 3 March 2003 • JChemEd.chem.wisc.edu
In the Laboratory
Instrumentation Needed Computer with a data acquisition board and LabVIEW installed
analyze data (5). In this experiment, data were collected by using a Power Macintosh 8100/100 desktop computer with a 12-bit analog-to-digital converter (National Instruments).
High-voltage power supply that can deliver 0.3–1 kV
Experimental Details
Low-voltage power supply or two 9-V batteries
Reagents All reagents were ACS grade chemicals from Sigma. The buffer used for all experiments was 10 mM sodium borate, pH 7.5, adjusted with a 5.00 N HCl solution. The analytes were a mixture of two dyes: Evans Blue and Night Blue. Stock solutions of 0.15 M of each dye were made. The stock solutions were mixed to make a 15 mM solution used for electrophoresis. A 10 mM NaOH solution was made to rinse the channels of the microchip. Nanopure water was used to make all solutions.
Vertical mill with diamond-embedded drillers or a digital cutter Furnace with a ±5 ⬚C precision
Micromachining A Supermax Vertical Mill (Yeong Chin Machinery Industries Co., LTD, Taiwan) was used to etch the channels on a 75-mm long × 25-mm wide microscope glass slide (Ward’s Natural Science Establishment, Monterrey, CA). The design of the channels was a double T-shape (Figure 2). The channels were drilled with diamond-embedded drillers. Wells with 4-mm diameters were drilled at the ends of the channels. A cover slide was prepared by drilling four holes to have access to the wells. The etched slide and cover slide were cleaned and placed in a 0.20 M H2O2 solution for about 30 min; then they were assembled together with clamps. The assembled slides were placed in a furnace at 540 ⬚C for 2 h so that lamination could begin. The temperature was raised to 610 ⬚C for 4 h and then lowered to 540 ⬚C for the last 2 h of the annealing process. The finished etch and laminated slides, or “microchip”, were allowed to cool over night. Electronic Circuit Design The electronic portion of this experiment permitted the construction of an effective system at a very low cost (about $40). The lamp consisted of a red high-intensity LED (Linrose Electronics Inc., Plainview, NY). The use of a highintensity LED provided sufficient intensity to allow detection on a microchip using a photosensitive chip (Opt 301, Burr Brown Corp., http://www.Burr-Brown.com, accessed Oct 2002). Restriction of the light path to the channel of the microchip was achieved by using optical fibers. The diameters of the fibers were approximately the same as the channel’s diameter. A slit, which closely matched the channel’s diameter, was constructed under the microchip’s channel with the use of black tape. In order to compensate for light variations and to increase the signal-to-noise ratio, a reference beam was implemented. This was achieved by creating an aluminum holder for the LED, which served as a beam splitter and can be placed on the side or top of the cover. This holder had a rectangular shape on the outside and forked on the inside (4). It divided the light of the LED into two beams; each was detected by a separate photodiode. One beam was directed at the reference photodiode and the other to the separation channel. The processing of the two resulting detector outputs was achieved with a difference amplifier. After alignment, the optical fibers and holder were glued together permanently. The design of the detector circuitry is shown in Figure 3. Data Acquisition LabVIEW 5.0 (National Instruments, Austin, TX) was used for data acquisition. LabVIEW programs have been successfully implemented in chemistry laboratories to sample and
high-intesity LED optic fibers high-voltage input
etched-glass slide
photodiodes
signal output
computer + LabVIEW
Figure 1. Simplified diagram of the microscale capillary electrophoresis instrument. The LED was placed in an aluminum holder, which served as beam splitter. Two optical fibers divided and carried the light of the LED to the inside of the wooden box where the glass microchip and sensor were located.
waste
buffer
black tape placed under the microchip to create a slit detection buffer region
sample additional slit for the reference photodiode Figure 2. Layout of the 75-mm long × 25-mm wide glass microchip. The channels were 400-µm wide and 50-µm deep.
JChemEd.chem.wisc.edu • Vol. 80 No. 3 March 2003 • Journal of Chemical Education
317
In the Laboratory
Procedure Five L of a 15.0 mM dye solution were placed in the sample reservoir. Approximately 50 pL of this solution were injected electrokinetically. A high-voltage power supply was linked to four connectors (banana plugs) soldered to platinum wire. The wire led to each well on the microchip, which delivered the voltage for injection and separation. For injection, a potential of 300 V was applied to the waste reservoir with the sample reservoir held at ground. Once the dye got to the cross section of the channel, 1000 V are applied between the buffer reservoirs, and the voltage polarity at the
V+
V− 9V
9V
Opt 301 1M 47n
Hazards The volumes of dye needed for this experiment are very small (
