EEN 1042 Physical Electronics

FACULTY OF ENGINEERING

LAB SHEET

NANO/MICRO-ELECTROMECHANICAL SYSTEMS

(N/MEMS)

ENT4046

TRIMESTER II (2012/2013)

MS 1 : MEMS (Prediction)

MS 2 : MEMS (Experiment)

*Note: On-the-spot evaluation will be carried out during or at the end of the experiment based on preparatory questions. Students are advised to read through this lab sheet before doing experiment. Your performance, teamwork effort, and learning attitude will count towards the marks.

MEMS: Microchip electrophoresis with capacitively-coupled contactless conductivity detection

Objectives

After completing the two laboratory phases on microchip electrophoresis (MCE) with capacitively-coupled contactless conductivity detection (C4D):

MS 1 : MEMS (Prediction)

MS 2 : MEMS (Experiment)

the students will be able

(1)  To understand the operation principle of microchip electrophoresis, a microfluidic-based MEMS application.

(2)  To perform experimental studies and analysis with contactless conductivity measurement in electrophoresis process.

Introduction

Microfluidic is a distinct part of MEMS that utilized the laminar flow of fluid in micro-scaled channels or capillaries. In contrast of the chaotic and unpredictable nature of turbulent flow, position of a particle in the fluid stream as a function of time can be determined in laminar flow by taking into account the diffusion process and the effects of external forces. One of the applications based on laminar flow in microfluidic devices is microchip electrophoresis, where the process of electrophoresis is performed on a single chip for injection, separation, transportation and characterization. The configuration has the advantages of being smaller in sample volume, minimal use of reagent, higher in applied field, higher velocities and thus faster to produce results.

Electrophoresis is the process where the motion of a charged (ions) caused by a non‐uniform electric field. Figure 1 show an example of capillary electrophoresis. The velocity of an ion of charge, in a homogeneous medium, is proportional to the applied electric field. Therefore, ions will move at characteristic velocities determined by their charges, radii, and mobilities.

Figure 1 Capillary Electrophoresis with optical detection.

When an electric field E is applied to a spherical particle of charge Ze and radius a in a stationary liquid of low electrical conductivity, say de-ionized water, particle movement is influenced by the applied electric field. The low conductivity of the liquid implies the lack of ions that otherwise would have accumulated around the charged particle and partly neutralized its charge (electrical screening). Figure 2 show the forces that are acting on the charged particle.

Figure 2. Electrophoresis. A spherical particle of charge Ze and radius a moves in a low-conductivity liquid with viscosity h under the infuence of an applied electrical Field E. The motion becomes stationary at the velocity uep, when the Stokes drag force Fdrag balances the electrical driving force Fel.

The electric force is simply Fel=ZeE. During a short time-scale of a few µs, the charged particle reaches steady-state motion, where the electrophoretic velocity uep, is due to viscous drag force. In this situation the Stokes drag force Fdrag=-6phauep, balances Fel,

Ftotal=Fel+Fdrag=0 (1)

uep=Ze6pha E=μionE (2)

where the proportionality constant μion=Ze6pha is called the ionic or electrophoretic mobility, (-ve for anions) (cm2·(V·s)-1)

uep is the electrophoretic velocity or migration rate (cm·s-1),

E is the electric field (V·cm-1),

h is the viscosity of the medium (Pa. s)

The electrophoretic mobility is proportional to the ionic charge of a sample and inversely proportional to any frictional forces present in the buffer. When two species in a sample have different charges or experience different frictional forces, they will separate from one another as they migrate through a buffer solution. The frictional forces experienced by an analyte ion depend on viscosity of the medium and the size and shape of the ion.

a= kBT6phD (3)

where kB is the Boltzmann constant, and T is the temperature, D is the diffusion coefficient.

The electrophoretic mobility can be determined experimentally from the migration time and the field strength:

μion=(Ltr)(LtV) (4)

where L is the distance from the inlet to the detection point, tr is the time required for the analyte to reach the detection point (migration time), V is the applied voltage (field strength), and Lt is the total length of the capillary. The experimental values for some ions are shown in Table 1. In addition, the ionic mobility is directly related to the ionic conductivity σion

σion=Zecionμion (5)

where cion is the ionic concentration (in M). Since only charged ions are affected by the electric field, neutral analytes are poorly separated by capillary electrophoresis. (1 mM is 100 mol/m3, 1 mol contains NA or 6.02214 ×1023 atoms or molecules)

Table 1. Experimental values for ionic mobility and diffusivity for small ions in aqueous

solutions at small concentrations.

The velocity of migration of an analyte in capillary electrophoresis will also depend upon the rate of electroosmotic flow (EOF) of the buffer solution. In a typical system, the electro-osmotic flow is directed toward the negatively charged cathode so that the buffer flows through the capillary from the source vial to the destination vial. Separated by differing electrophoretic mobilities, analytes migrate toward the electrode of opposite charge. As a result, negatively charged analytes are attracted to the positively charged anode, counter to the EOF, while positively charged analytes are attracted to the cathode. The velocity of the electroosmotic flow is given by uo=μoE.

After injection and separation, analytes in electrophoresis process can be detected by various methods, namely optical based such laser induced fluorescence or absorption, mass spectroscopy, electrochemical detection and also contactless conductivity detection. In the current experiment, the conductivity of the fluid is measured by electrodes and reference electrodes configured on the microchip. The principle of measurement can also be used in capillary electrophoresis, Ion Chromatography (IC) and High-Performance Liquid Chromatography (HPLC) and Flow Injection Analysis (FIA). The basis of contactless conductivity measurements is explained in Figure 3.

Fig 3 (a) C4D uses a transmitter electrode to subject a sample region to a large amplitude, high frequency electromagnetic signal. A corresponding, attenuated, AC signal registers at a receiver electrode. (b) The size of the received signal is affected by the conductivity of the sample. (c) The received AC signal is deconvoluted to convert the amplitude into a DC analog voltage signal appropriate for data collection.

In capactively coupled contactless conductivity measurements an AC-voltage is applied to one of the galvanically isolated electrodes and the resulting AC-current is measured at the second electrode. This is possible as the electrodes form capacitors with the electrolyte solution which are transparent for ac-signals.

The usual axial arrangement of two tubular electrodes used on a conventional capillary is illustrated in Fig. 4(a) along with a simplified equivalent circuit diagram given in Fig. 4(b). The method has been widely applied in the analysis of inorganic ions, organic ions, and bio-molecules. An example for measurement of inorganic cation in blood sample is shown in Figure 5.

Fig. 4 (a) Schematic drawing of a C4D cell for conventional capillary electro-phoresis. (b) Simplified equivalent circuit diagram in presence of direct capacitive coupling between the electrodes. (c) Schematic drawing of contactless conductivity detection for microchip electrophoresis.

Fig. 5 Determination of inorganic cations in blood serum sample using MCE-C4D . Electrolyte solution, 15 mM L-arginine, 10.90 mM maleic acid, 1.5 mM 18-crown-6 (pH 5.85). (A) Blood serum sample (diluted 1:50), (B) blood serum sample spiked with 1.5 mM Li1 (diluted 1:50).

MS1 Prediction

Microchip electrophoresis with C4D

Activities:

1.  Identify the parameters that contribute to ions movement in solution.

2.  Detection of cation and anion in test solution.

3.  Find the appropriate concentration to be used in microchip electrophoresis experiment by varying the concentration of background electrolyte or analytes (cation and anions).

Test solution:

Background electrolytes (BGE) = 0.5 M acetic acid

Sample = 1 mM LiCl, KNO3, Na2SO4 in deionised water

Micronit microfluidic chip T35100C4D (Appendix A)

Software: Peakmaster 5.3 with database of constituents by Takeshi Hirokawa (http://web.natur.cuni.cz/gas/)

Procedure

1.  Understand and identify the channel dimension and length of channel to detector with reference to Micronit microfluidic chip (Appendix A)

2.  Fixed an applied voltage for the test solution. *note the maximum voltage for ER 230 is 3000 V.

3.  Varied the concentration of the background constituent (BGE) from 100 mM to 1000 M. Choose Li+ , K+ and Na+ for cation and HCl, H2SO4 and HNO3 for anion analysis. Observed the changes. *note that at least 5 points are needed

4.  Recorded and export the signal level and separation time for cation, plot the electropherograms of all concentration in a graph (for example a 3D graph). Repeat the same for anion.

5.  Choose a concentration of BGE to be used later for cation analysis in MS2 experiment.

6.  Varied the voltage from 500 to 2000 V with 500 V increment.

7.  Record the signal level and separation time versus applied electric field. (The results will be compared to the electropherogram from experimental measurement).

8.  Find the electrophoretic velocity and electrophoretic mobility of the cation from the prediction’s results.

9.  Discuss the observations, which include parameters affecting ions movements and characteristics of the electropherogram.

Reference: example results of the test solution

Results for Cations

The electropherogram for the cations, run at 100 μM concentration, is shown in Figure P1. As predicted by Peakmaster software, three negative peaks are observed. The peaks are negative because the three cations Li+, K+ and Na+ are less conductive than the H+ cation they are displacing in the background electrolyte. The electropherogram can be recorded with positive peaks by selecting Invert in the C4D Amplifier window, in the Hardware Settings of the PowerChrom software. Figure P1 shows the data with a sample injection time of 20 seconds. Increasing the injection time, to 30 and 40 seconds, resulted in larger peaks but the separation of the peaks was not as good.

Fig. P1 Electropherogram for cation

Results for Anions

Figure P2 shows the electropherogram for anion analysed at 1 mM. This concentration will overload the system, resulting in one large peak which cannot be resolved.

The solution for running anions should be diluted to 100 μM with deionised water. The electropherogram is shown in Figure P2b. Peakmaster software predicts the two positive peaks obtained, where the HSO4- and NO3- peaks cannot be resolved. Separating the anions under these conditions will not produce an EOF peak, because the EOF travels away from the detector, towards Reservoir 2

.

Fig. P2 The effect of concentration on the measured signal in electropherogram.

MS2 MEMS (Experiment)

Microchip electrophoresis with C4D

The analysis of the EC20 Standard Test Solutions by microchip electrophoresis with capacitively-coupled contactless conductivity detection (MCE-C4D ) will be performed.

Activities:

1.  Based on the test solution provided, perform experiment of microchip electrophoresis with capacitively-coupled contactless conductivity detection for cations.

2.  Investigate the effects such as injection time and electric field (< 2000 V) on the test sample.

3.  Obtain the electrophoretic mobility and conductivity of the test solution experimentally.

4.  Report the experimental findings, which include: explanation of how injection, separation and contactless conductivity measurement are achieved, the effects of parameters affecting ion movements, difficulties faced in an actual measurement, conclude on the method and findings in the experiment.

Injection, separation and detection

This section describes a procedure for a microchip electrophoresis experiment using a floating injection. This type of injection requires control of the voltages at only two of the fours reservoirs of the chip. Thus only one High Voltage Sequencer is needed.

During the floating injection step, 1000 V is applied across the chip (between reservoirs 1 and 3) (Figure E1 and more details in Appendix A).The channels of the chip are in a double-T configuration, and this voltage fills the small area between the offset of the channels. During the separation step, 1000 V is applied along the chip (between reservoirs 2 and 4). This moves the sample towards the detector and reservoir 4, separating the ions as they travel.

A gated injection can be performed with the control of the voltage at all four reservoirs of the chip (and thus needs two High Voltage Sequencers).

Fig. E1 Microchip layout

1.  Equipment Required

ER255 or ER455 Microfluidic Chip Electrophoresis Kit (Fig. E2), including:

ER225 C4D Data System

ET225 Micronit Chip Electrophoresis Platform with C4D headstage

ER230 HV Sequencer (one or two units)

ET145-4 CE Microchip (45 mm) kit

PowerChrom and Sequencer software

EC20 Standard Test solutions

Background Electrolyte (BGE) = 0.5 M acetic acid

Sample = 1mM LiCl, KNO3, Na2SO4 in deionised water

One 20 - 200 μL pipette, with pipette tips

One syringe 5 mL

Deionised water

Lint-free tissues