BIS method is based on real time monitoring of target analyte as it is being accumulated on beads (A), held within a flow cell. The key component of the instrument is the lab-on-valve module (B) and the microfluidic manipulations are carried out either in Sequential Injection or programmable Flow Injection mode.
Design of the flow cell is a complex issue since the following conditions must be met:
Bead Injection Spectrophotometry
In Radial Flow Cell Configuration
3.4.1.
- the beads must be retained in the light path
- the beads must be packed in a well-defined geometry
- the light beam must interrogate the majority of beads
- amount of beads situated upstream from the beam must be minimized
- at the end of measuring cycle all beads must be swept clear from the flow cell cavity
- the beads must be sufficiently transparent in order to allow monitoring by spectrophotometry4
These conditions are met by the flow cell design that uses 600µm quartz fibers, clad by steel or PEEK tubing. The gap (2 mm wide) between the fibers defines the optical path that is perpendicular to the flow channel 1.6 mm wide, leading from port #2 through the cell, the outlet of which is blocked by a retaining plug. The plug has an O.D. 30 µm smaller than the I.D. of the channel, while the I.D. of the channel within the plug is 100 µm. These dimensions allow the carrier stream to pass through and alongside the plug, while the beads are retained. The quantity of beads (typically 2µL of packed bed) is defined by the volume of injected bead suspension. Flow programming is the essential component of this design: beads are packed at a flow rate of 50µL/sec, perfused by analyte at 1µL/sec and discarded at a flow rate of 200µL/sec.
As to microfluidic manipulations key to successful performance of BI lies in the design of reliable
- bead transport
- bead packing because it influences reproducibility in which bead layer is perfused
- reproducible metering of beads so that variation of the amount of beads retimed in flow cell is minimized
While for renewable column chromatography it is sufficient to meet the first two requirements, BIS is more difficult to perform because the volume of bead suspension and packing density of the bead layer in the flow cell must be very precisely controlled.
