Chapter 5 Embedded HPLC System
5.4 Laser-Induced Fluorescence Detection
has a square-like column cross-section does have lower porosity which means particle packing is denser in the embedded column under the same particle packing pressure.
0 30 60 90 120 150 180
0.0 0.1 0.2 0.3 0.4 0.5
Linear fitting (50 to 160 psi) Y =-0.0448+0.00295 X
Flow rate (
µL/ min )
Pressure (psi)
5µm-C18 porous particles Column packed at 200 psi Column: 22mm x 50µm x 65 µm Fluid: DI water at 25 oC
Viscosity = 8.9 mPoiseuille 50µm x 65µm x 22mm
Figure 5-12: Flow rate vs. pressure curve of the embedded LC column. Linear fitting was done to the higher pressure (> 50 psi) data points.
The excitation light sources for fluorescence detection are versatile, from mercury vapor to xenon to quartz halogen to deuterium to lasers. Using a laser as the excitation source for fluorescence detection is called Laser-Induced Fluorescence (LIF) detection.
Lasers have well-defined wavelengths and can be focused on a very small area, yielding effective cell volumes as small as 1 nl [5]. Therefore, LIF has extremely high sensitivity and it can be easily incorporated to microchip HPLC system using off-chip optics [6-8].
The transparency characteristics of parylene to visible light make LIF a favorable detection method for our microchip HPLC system. Besides, due to the embedded-type channel configuration, incident laser light will scatter vigorously inside the channel from the roughened silicon surface (roughened by DRIE etching) and results in a thorough excitation inside the detection cell, which in turn produces stronger fluorescence signal.
While it is true that a stronger reflective laser signal will be generated due to this scattering phenomenon, the laser signal level will be filtered by a bandpass filter to a negligible level and therefore the overall SNR (signal-to-noise ratio) will be enhanced by using this embedded-type channel. This advantage is not available when an anchored- type channel is used. Finally, the scattering phenomenon also provides information regarding laser spot alignment to the embedded LIF cell. When best alignment is achieved, scattering is strongest and so will be the signal picked up by the photodiode (bandpass filter is removed from the optical path during the alignment).
5.4.2 System Design and Fabrication
Figure 5-13 shows the schematic plot and the real setup of our homemade LIF system. All optics and optical paths are lying on the XY plane instead of stacking up in
the Z direction so as to facilitate the optics alignment procedure as well as the integration of microchip HPLC components. The excitation light source is a 488 nm argon ion laser with adjustable output power (National Laser Company, Salt Lake city, Utah). The emitted laser passes though a homemade, 200-µm-sized pinhole chip followed by a stepped power density filter. The laser beam is then reflected by a visible-laser quality mirror followed by a laser-line bandpass filter (bandwidth equal to 1.9 nm with a central wavelength of 488 nm). The laser beam is then focused using a simple lens (diameter = 40 mm, effective focal length = 100 mm) onto the LIF cell of the microchip with an incident angle of 60 degrees. The reflected laser beam is absorbed by a laser beam stopper. A microscope objective (50X, NA = 0.45) is used to collect the fluorescence emitted from the LIF cell. Light collected by the objective then passes through a longpass filter (transition width < 4.9 nm, edge steepness = 2.5 nm) to remove the 488 nm laser light source. Fluorescence signal is then collected by the photodide (DET110, Thorlabs, North Newton, NJ) and a current signal is accordingly generated. The current signal is converted to a voltage signal using a simple Op-Amp circuit. The voltage signal is then picked up by the data acquisition unit (HP34970A) and is sent to a PC where the chromatogram is recorded. Most system components, including the laser, pinhole, mirror, lens, microchip/jig, objective, and photodiode are fixed on top of XYZ stages to allow maximum alignment flexibility. The microchip/jig piece is fixed to the XYZ stage with screws to avoid movements caused by the pulling of fluidic tubing from jig backside.
In order to align the laser spot onto the LIF cell of the microchip, a CCD camera, a foldable mirror, and a monitor were assembled next to the objective to catch the chip surface image around the LIF cell. After the laser spot alignment is done, the mirror is
folded down to switch the optical path back to the longpass filter. Figure 5-14 shows an example of a successfully aligned laser spot on the LIF cell.
488nm Argon ion laser
Power filter Mirror
BP filter
Lens
Chip/Jig Laser absorber Objective
Foldable mirror CCD
camera
LP filter
Photo diode Monitor
Amp circuit
Data acquisition unit
PC Pinhole
Sample pump
Mobile phase pump
Multimeter
Universal source
(a)
(b)
Figure 5-13: (a) Schematic plot of the LIF system, (b) pictures of the actual system.
Light-absorbing black boards were used to isolate optical paths from each other to reduce noise from laser light scattering. The whole LIF detection system was built on top of an anti-vibration table and was shielded from ambient light and dust using black rubberized fabric.
50 µm
Figure 5-14: Laser spot alignment to microchip LIF detection cell.
5.4.3 LIF Detection Characterization
Background noise level of the LIF detection system was examined. Fluorescein solution samples with different concentrations were injected into our embedded HPLC microchip where no stationary-phase particles were packed. A 1.5 mW/488 nm laser spot was focused on the LIF cell, which has a cell volume of 0.5 nL, to excite fluorescein molecules. An Op-Amp circuit with an amplification resistance of 66 MΩ was used to convert photodiode output current into a voltage signal. Signal was sampled at a frequency of 10 Hz and signal standard deviation was calculated and plotted versus signal as shown in Figure 5-15. LIF signal increased linearly with the fluorescein concentration in the sample. The limit of detection (LOD) of the system was calculated to be 125 nM with an SNR of 10. One should note 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 5-15: LIF signal standard deviation vs. signal level.