Whole Column Resistance Detection for Focusing Methods of Separation
Original Publication Date: 2004-May-11
Included in the Prior Art Database: 2004-May-11
National Institute of Standards and Technology
This disclosure describes a very simple detection scheme for use with analyte focusing methods such as electric field gradient focusing (EFGF), temperature gradient focusing (TGF), micellar affinity gradient focusing (MAGF), and isoelectric focusing (IEF). For each of these focusing methods, a high voltage is applied to the separation column to cause the analytes in the column to move towards a point of zero velocity, where each analyte is focused into a peak. Most commonly, many peaks are focused at once and each peak is detected using an external detection scheme such as laser induced fluorescence (LIF), ultra-violet (UV) absorbance, or mass spectrometry. For the detection scheme described here, the focusing method is adjusted so that only one peak is focused at a time (or so that only one peak is focused in a sensitive area at a time), and the detection simply consists of measuring the resistance of the entire separation column. In its simplest embodiment, the detection scheme described here is implemented merely by measuring the current flow that results from the application of the high voltage to the column. When a peak of analyte is focused, it locally changes the resistivity of the solution in the column resulting in a change in the total column resistance that can be easily measured. In a typical measurement with this type of detection, the focusing conditions are varied over the course of a separation so that the zero-velocity point for each analyte is scanned across the gradient focusing region, and each analyte is thus in turn, sequentially injected, focused (and detected), and then defocused and eluted from the column.