Measurement of Detector Linearity and Effective Pathlength in Capillary Electrophoresis

www.lcgceurope.com Introduction The quality of photometric detection in capillary electrophoresis (CE) can be characterized by detector sensitivity and linearity. From the Beer-Lambert law it follows that the detector sensitivity is directly proportional to the detection pathlength. However, this might not be easy to estimate with cells with geometries that result in a range of possible pathlengths. In such instances, it is the effective pathlength (i.e., the pathlength of an equivalent rectangular cell) that is most important. Consider the situation illustrated in Figure 1. A ray of light that passes exactly through the centre of the capillary (R1) will have a pathlength equal to the inner diameter (i.d.) of the capillary, whilst a ray of light that passes through the capillary slightly off-centre (R2) will have a pathlength slightly less than the capillary i.d. The pathlength will become progressively shorter as the ray of light passes through the capillary further from the centre (R3). Depending on the design of the optics it is also possible for non-parallel light beams to pass through the capillary (R4). The individual pathlengths will combine to produce an effective pathlength, which ideally would be equal to (but in reality will always be less than) the capillary i.d. Bruin et al.1 have presented a geometrical mathematical model for calculation of the effective pathlength for parallel beams of light passing through a cylindrical capillary. While this might provide a rough approximation of effective pathlength (depending on the optics design) it is not generally applicable to on-capillary detectors such as those used in CE because parallel light rays are unlikely Measurement of Detector Linearity and Effective Pathlength in Capillary Electrophoresis