Temperature-Controlled Microchip Liquid Chromatography System

92 Figure 4-13: FEMLAB simulation showing an exaggerated vertical displacement of thermally isolated silicon island loaded with uniform pressure from below. 93 Figure 4-14: FEMLAB simulation showing vertical displacement of thermally isolated silicon island as a function of DRIE channel depth "d".

MEMS Technology

Introduction

MEMS by Definition
MEMS Fabrication Technology

Bulk Micromachining
Surface Micromachining
Other Technologies
Summary

Market and Products of MEMS Technology

The fabrication technique uses wet (chemical) or dry (plasma, gas) etching to etch the substrate with masking films to form microstructures in the substrate. At the beginning of the process, a sacrificial layer is deposited and patterned on the substrate (Figure 1-3).

Figure 1-1: Bulk micromachining technology. (a) Anisotropic etching on <100> surface orientation, and (b) anisotropic etching on <110> surface orientation

Lab-on-a-Chip System by MEMS Technology

Applications of Lab-on-a-Chip System
Challenges and Outlooks

On-chip sample preparation proves to be an extremely challenging task for all LOC systems. Therefore, a robust, low-volume world-to-chip interface technology needs to be developed [37].

Parylene as a Microstructure Material

Introduction to Parylene
Applications of Parylene Thin Film in MEMS

Furthermore, parylene is biocompatible (USP Class VI), which makes it a good candidate material for the production of long-lasting human implants or biomimetic components. In terms of optical properties, parylene is quite transparent in the visible light range.

Figure 1-8: Parylene deposition system and the involved chemical processes.

Bibliography

Tai, "Surface micromachined leakage proof parylene check valve," præsenteret på den 14. IEEE International Conference on Micro Electro Mechanical Systems (MEMS 2001), Interlaken, Schweiz, 2001. Tai, "Integrated surface-micromachined masseflow controller," præsenteret på 16. IEEE International Conference on Micro Electro Mechanical Systems (MEMS 2003), Kyoto, Japan, 2003.

Evolution of HPLC Technology

High-Performance Liquid Chromatography

Introduction
History
Theory

Analyte Retention and Chromatogram
Height Equivalent to a Theoretical Plate
Reduced/Dimensionless Parameters
Band-Broadening Phenomenon

Sample molecules adsorb to the stationary phase in the initial part of the column and form a narrow sample band. The retention factor of the later eluting analyte is in the numerator, so the value is always greater than one.

Figure 2-1: Illustration of the chromatographic separation process.

HPLC Instrumentation

Separation Column

Separation Column Tube
Separation Column Packing Material

Solvent Pumps
Sample Injection Valves
Analyte Detectors

Particle pore size is the average dimension of the flow channels within the porous particle. Constant flow rate pumps are most desirable for repeatable retention times, but these pumps often produce noise in the detector due to the cyclic nature of their pumping action.

Figure 2-8: A six-port, two-position manual sample injection valve.

Logical Trend of HPLC Instrumentation

Predictions from the Theory
Benefits of Microchip HPLC System

An overall miniaturized HPLC system will be portable and therefore many critical applications are possible, such as point-of-use HPLC analysis or portable systems for personal health diagnostics. It is conceivable that the need for HPLC miniaturization will lead to the realization of the construction of a complete HPLC system on a single chip. By integrating all HPLC components on a single chip, connector wipe/dead volume can be virtually eliminated.

Another advantage of the lab-on-a-chip HPLC system is its potentially much lower costs.

Review of Microchip HPLC Systems

The design concept of the anchored-type parylene channel is to mechanically anchor the upper parylene layer to the silicon substrate. Peak assignments in the chromatogram were based on the hydrophobicity ranking of the derivatized amino acids [28]. To confirm that model molecules indeed play a role in forming the self-assembling compound, that is, the nanoparticles with model molecules are linked together, the electrical property of the compound was studied.

Indeed, as stated in the title of Richard Feynman's famous speech, "There's plenty of room at the bottom," the field of HPLC microarray research is young, exciting, and full of opportunity.

Table 2-2: Comparisons between LC and CE [35-37].

Comparisons bewteen LC and CE

Conclusions

The pressure capacity of the embedded parylene channel has been tested to over 1000 psi without breaking [18]. Based on the experimental data of the TCR (temperature coefficient of resistance) of the platinum heater, as shown in Figure 4-5, the temperature of the heater can be derived from the resistance of the heater. The pressure capacity of the embedded parylene channel type has been tested in excess of 1000 psi without breaking.

Using those component values, HSPICE analysis of the circuit, as shown in Figure 5-21(a), was performed.

Bibliography

High-Pressure Microfluidic Channel Technology

Introduction
Review of Microfluidic Channel Technology…

PDMS Microfluidic Channel
Thermal-Bonding Microfluidic Channel
Nitride Microfluidic Channel
Parylene Microfluidic Channel
Buried Microfluidic Channel

High-Pressure Parylene Microfluidic Channel Technology

Anchored-Type Parylene Microfluidic Channel
Embedded-Type Parylene Microfluidic Channel
Comparisons

Conclusions
Bibliography

In general, the thermally bonding microfluidic channel has a large pressure capacity (bond strengths up to 20 MPa or approximately 3,000 psi have been reported [11]). The parylene microfluidic channel is created by first depositing a parylene layer using a room temperature CVD system (Specialty Coating Systems, Indianapolis, IN) on top of a silicon or glass substrate to form the bottom wall of the channel. Buried microfluidic channels or buried channel technology (BCT) have been developed and proposed for various biochemical applications [15].

Thus, we have developed and illustrated two types of novel high-pressure parylene microfluidic channel technologies in this chapter, that is, anchored type and embedded type.

Figure 3-1: Pictures of assorted microfluidic channels. (a) PDMS stamp that shows channel topography, (b) silicon/silicon thermal-bonding channel, (c) nitride channel, (d) parylene channel/nozzle, (e) buried channel [15]

Temperature-Controlled Microchip HPLC System

Temperature Gradient Interaction Chromatography

On the other hand, it has been found that a temporal temperature gradient (i.e. changing the temperature of the LC column as a function of time) can be as effective as a solvent gradient in terms of modulating the elution power of the mobile phase. The use of a temperature gradient to achieve or improve analyte separation is categorized as temperature gradient interaction chromatography (TGIC) [3-10]. Now that the chromatogram is transformed with a time-temperature gradient (Figure 4-1 (b)), early peaks can be better separated at low temperature due to longer retention times and differences in retention times between peaks.

Compared to a solvent gradient, a temperature gradient is much easier to perform on a microchip HPLC system.

Figure 4-1: Working principles of TGIC. (a) Isothermal elution where temperature is fixed throughout the elution process

Chip Design, Fabrication, and, Characterization

Low-Power, Low-Pressure-Capacity System

Design and Fabrication
System Characterization

High-Power, High-Pressure-Capacity System

Application of Parylene Thermal Isolation Technology

DRIE is performed on the wafer back again to etch through the silicon wafer for liquid access channels and thermal isolation of air gaps. Results show that heater power consumption is reduced by 58% when the column is surrounded by the air gap thermal insulation structure (Figure 4-11). A FEMLAB simulation was used to study the mechanical stiffness of the parylene-reinforced thermal insulation structure.

Using our thermal isolation technology, three temperature zones defined by air gaps were created on the silicon substrate, as shown in Figure 4-17.

Figure 4-2: Process flow for the low-power, low-pressure-capacity TGIC microchip.

TGIC Chip Packaging

Stationary-Phase Particle Packing

The tubing is then connected to the microfluidic port of the gyro at one end and the other end is connected to a 200 psi filtered nitrogen gas pressure source. The slurry is then injected into the column where the particles are stopped by the 4 μm high filter structure and begin to pack into the column as shown in Figure 4-20.

Chip Temperature Programming

Examples of Separation

Amino Acid Separation

Introduction
Sample Preparation
Electrochemical Detection
Separation Results and Comments

Low Density Lipoprotein Separation

Introduction
Sample Preparation
Conductivity Detection
Separation Results and Comments

Typical cyclic voltammetry of a derivatized amino acid at 23 0C. From this CV for ALA, a peak current potential of 0.62 V was obtained. The high-pressure capability HPLC microchips presented in 4.2.2 were used in the amino acid separation assay. When the amino acid peak passed through the electrochemical sensor, the resistance of the sensor decreased due to the electrochemical reaction.

The electrochemical sensor used for amino acid sensing in 4.6.1 is used here as a conductivity sensor for LDL.

Figure 4-22: Cyclic voltammetry characterization of the derivatized amino acids. (a) Typical cyclic voltammetry of derivatized amino acid at 23 0 C

Conclusions

Bibliography

Tai, "On-chip temperature gradient liquid chromatography", presented at the 18th IEEE International Conference on Micro Electro Mechanical Systems (MEMS 2005), Miami, USA, 2005. Lee, "Ion liquid chromatography on a chip with bead-packed parylene column", presented at the 17th IEEE International Conference on Micro Electro Mechanical Systems (MEMS 2004), Maastricht, Netherlands, 2004. Tai, "Robust parylene-to-silicon mechanical anchoring," presented at the 16th IEEE International Conference on Micro Electromechanical Systems (MEMS2003), Kyoto, Japan, 2003.

Tai, “Parylene-enhanced thermal isolation technology for microfluidic system-on-chip applications,” presented at the 13th International Conference on Solid-State Sensors, Actuators and Microsystems, Seoul, Korea, 2005.

Embedded HPLC System

Introduction

Differing in manufacturing complexity and device functionality, two versions of embedded systems are proposed, the single-mesh system and the multi-mesh system. Various detection methods, including LIF detection and capacitively coupled contactless conductivity detection (C4D), have been investigated for use with the proposed embedded systems.

Single-Mask Embedded HPLC System

Therefore, smaller oxide aperture around the filter section, compared to column section, results in a smaller XeF2 etched channel cross-section. Second, after oxide opening around the filter section is sealed by parylene deposition, filter cross-section continues to shrink due to the parylene coating to its inner surface through the neighboring oxide opening in the column section. Due to the single-mask process criterion, no sophisticated on-chip sensor can be fabricated on this microchip HPLC system as shown in Figure 5-4.

However, LIF detection can be applied using off-chip optics (will be discussed more in 5.4).

Figure 5-1: Top view of the 15 µm parylene coating process. (a, b) Filter/column section, (c, d) liquid inlet/outlet before and after parylene coating

Multiple-Mask Embedded HPLC system

A dummy filter structure with a larger opening in the second parylene layer is placed over the channel to the working filter, as shown in step 1. Additionally, due to the highly planar chip topography, a direct-top clamping procedure using a glass plate can be easily accomplished. applied to increase duct pressure capacity to thousands of psi, as shown in Figure 5-11(c). The leakage rate under a certain pressure was obtained by measuring the moving speed of the liquid front in a Teflon tube connected to the column inlet.

The flow rate under a certain pressure was obtained by measuring the velocity of the liquid front in a Teflon tube connected to the column inlet.

Figure 5-7: Fabrication process flow for the multiple-mask embedded HPLC system.

Laser-Induced Fluorescence Detection

Introduciton
System Design and Fabrication
LIF Detection Characterization

The laser beam is then focused by a simple lens (diameter = 40 mm, effective focal length = 100 mm) onto the LIF microchip cell with an incidence angle of 60 degrees. To align the laser spot on the LIF microchip cell, a CCD camera, folding mirror and monitor were assembled in addition to the lens to capture an image of the chip surface around the LIF cell. A 1.5 mW/488 nm laser spot was aimed at the LIF cell, which has a cell volume of 0.5 nL, to excite the fluorescein molecules.

It should be noted that the LOD of the LIF system can be improved by more than three orders of magnitude by replacing the photodiode with a photomultiplier tube (PMT) [6].

Figure 5-13: (a) Schematic plot of the LIF system, (b) pictures of the actual system.

Capacitively-Coupled Contactless Conductivity Detection

Sensor Design and Fabrication
Sensor Characterization
Resonance-Induced Sensitivity Enhancement for C 4 D

Therefore, it can be expected that microchip C4D sensitivity will be much lower than capillary C4D. We will show that at this resonant frequency the C4D sensitivity is dramatically improved by more than 10,000 times. It is found that the resonant frequency remains at 930.23 kHz for a solution resistance ranging from 1 kΩ to 100 kΩ and increases slightly to 930.47 kHz when the solution resistance is 1 MΩ.

In this case, using RISE will remove the double layer capacitance on top of the electrodes and the parasitic capacitance from the circuit.

Figure 5-16: C 4 D for capillary separation systems [11].

LABVIEW Program for Complete HPLC Procedure Control

Program Design
Program Performance Characterization

The second part of the program allows users to set pumping parameters for sample injection and elution procedures. The program was designed to automatically save the LIF detection data and the measured microchip temperature profile data into separate files. Real-time graphs of this data are also displayed in the software interface so that users can monitor the progress of the separation.

The ability of the program to maintain constant temperature at different temperature levels was also investigated.

Figure 5-25: User-interface of the developed LABVIEW program for the complete automatic control of HPLC procedures, (top) temperature control panel, (bottom) pumping control panel

Examples of Separation

Daunorubicin Elution
Separation of Daunorubicin and Doxorubicin

The injected sample volume is controlled by the sample injection pump time and flow rate. In step 4, sample injection stops and mobile-phase injection begins to achieve sample elution and detection. Three identical elution procedures were performed and extremely good agreement between the three chromatograms including peak heights and retention times was obtained which means that the sample injection procedure as well as the column condition were quite robust.

Both compounds are used in pharmaceuticals as anticancer drugs, and both compounds have native fluorescence, so no derivatization process is required for sample preparation.

Figure 5-28: Sample injection and elution pumping procedures.

Conclusions

Bibliography

Belder, "Deep UV laser-induced fluorescence detection of unlabeled drugs and proteins in microarray electrophoresis," Analytical Chemistry, vol.

Packing Nanoparticles into HPLC Column

Introduction
Molecular-Self-Assembly-Assisted Nanoparticle Packing

Concept
System Design and Fabrication
Molecular-Self-Assembly Characterizations
Column Packing Results and Comments

Conclusions
Bibliography

The success of the proposed nanoparticle packing approach depends on the conjugation reaction between the thiol groups of the crosslinking molecules and the gold nanoparticles. In the assembly test phase, solvent A will be saturated with the model molecules at room temperature and solvent B will be used to resuspend the gold nanoparticles. The resistivity of the aggregate of pure gold nanoparticles is quite comparable to the resistivity of bulk gold, which is ~ 10-6 Ω-cm.

We investigated the size effect by using gold nanoparticles with different particle sizes (5 nm, 10 nm, and 30 nm) in the self-assembly experiments using the ethanol solvent system.

Figure 6-1: Micron-sized and nanometer-sized particles.

Conclusions

The aim of this thesis is to investigate the state-of-the-art technologies for the production of microchip HPLC systems and to study the chromatographic properties of such systems. A novel C4D sensor together with the RISE sensitivity enhancement method was first proposed and investigated for microarray HPLC analyte detection. Examining published works in the field of HPLC, future research on microarray HPLC can be divided into two categories.

The first category is to accelerate the commercialization of microarray HPLC systems, which requires further improvements in the integration of system components, separation efficiency (efficiency and reproducibility), and manufacturing costs.