Novel absorption detection techniques for capillary electrophoresis

Abstract

<p>Capillary electrophoresis (CE) has emerged as one of the most versatile separation methods. However, efficient separation is not sufficient unless coupled to adequate detection. The narrow inner diameter (I. D.) of the capillary column raises a big challenge to detection methods. For UV-vis absorption detection, the concentration sensitivity is only at the [mu]M level. Most commercial CE instruments are equipped with incoherent UV-vis lamps. Low-brightness, instability and inefficient coupling of the light source with the capillary limit the further improvement of UV-vis absorption detection in CE. The goals of this research have been to show the utility of laser-based absorption detection. Our approaches involve: (1) on-column double-beam laser absorption detection and its application to the detection of small ions and proteins, (2) absorption detection with the bubble-shaped flow cell;Section I presents double-beam laser absorption detection for capillary electrophoresis. It is based on the direct subtraction of reference and signal photocurrents with an electronic circuit (all-electronic canceller) to reduce background noise. The experimental results show that the noise-to-signal ratio in intensity is five times lower (1x10[superscript]-5 A.U.) than that of the best commercial CE system. Since there is also better light coupling with the capillary, a 25-fold improvement of the detection limit over commercial systems is achieved. This is the most sensitive direct absorption detection method to date for CE;Double-beam laser indirect absorption detection in CE is described in section II. By increasing the absorption pathlength and reducing noise through the all-electronic noise canceller, the performance of indirect absorption detection is substantially enhanced. The sensitivity is over 10 times better than that of the best commercial CE systems;Laser-based UV absorption in CE with vacuum photodiodes and the all-electronic canceller is discussed in section III. Despite the large inherent intensity noise in UV lasers, a 4-fold improvement in sensitivity is achieved compared to commercial CE systems. The main advantage is the longer optical pathlength due to the better light coupling with capillaries;A simple procedure for creating a bubble-shaped flow cell on a separation column is described in section IV. This cell can provide an extended pathlength for absorption detection in CE. With laser-based double beam detection, about an 8-fold enhancement in sensitivity is realized. The peak broadening in the bubble cell arises from the distorted flow due to the change in geometry. However, the electric field lines are still oriented axially throughout this region.</p>