Wavelength Modulated Back-Scatter Interferometry for Universal, On-Column Refractive Index Detection in Picoliter Volumes.

Abstract

Wavelength-modulated back scatter interferometry (M-BSI) is shown to improve the detection metrics for refractive index (RI) sensing in microseparations. In M-BSI, the output of a tunable diode laser is focused into the detection zone of a separation channel as the excitation wavelength is rapidly modulated. This spatially modulates the observed interference pattern, which is measured in the backscattered direction. Phase-sensitive detection using a split photodiode detector aligned on one fringe of the interference pattern is used to monitor RI changes as analytes are separated. Using sucrose standards, we report a detection limit of 700 μg/L in a 75 μm i.d. capillary at the 3σ level, corresponding to a detection volume of 90 pL. To validate the approach for electrophoretic separations, Na+ and Li+ were separated and detected with M-BSI and indirect-UV absorbance on the same capillary. A 4 mg/L NaCl and LiCl mixture leads to comparable separation efficiencies in the two detection schemes, with better signal-to-noise in the M-BSI detection, but less baseline stability. The latter arises in part from Joule heating, which influences RI measurements through the thermo-optic properties of the run buffer. To reduce this effect, a 25 μm i.d. capillary combined with active temperature control was used to detect the separation of sucrose, glucose, and lactose with M-BSI. The lack of suitable UV chromophores makes these analytes challenging to detect directly in ultrasmall volumes. Using a 55 mM NaOH run buffer, M-BSI is shown to detect the separation of a mixture of 174 mg/L sucrose, 97 mg/L glucose, and 172 mg/L lactose in a 15 pL detection volume. The universal on-column detection in ultrasmall volumes adds new capabilities for microanalysis platforms, while potentially reducing the footprint and costs of these systems.