Bioanalysis Using HSCE

We are developing HSCE with simultaneous BSI and fluorescence detection for serum protein analysis, immunoassays, and metabolic screening applications.

Direct Detection of Inorganic Ions and Underivatized Amino Acids in Seconds Using High-Speed Capillary Electrophoresis Coupled with Back-Scatter Interferometry

**(Analytical Methods, in press)**

High speed capillary electrophoresis (HSCE) combined with refractive index (RI) detection is developed for the rapid separation and detection of inorganic ions and amino acids. A mixture of three inorganic ions (K+, Na+, Li+) and eight amino acids (Lys, Arg, Ala, Gly, Val, Thr, Trp, Asp) are detected using back scatter interferometry (BSI), without the need for chemical modifications or contrast. A thin-walled separation capillary (50 mm i.d. by 80 mm o.d.) helps mitigate Joule heating at the high field strengths required for rapid separations. This, combined with a short 8 cm length-to-detector (10 cm total length), enables separations on the seconds time scale. Using a background electrolyte (BGE) of 4 M acetic acid (pH 1.6) and a field strength of 900 V/cm, all 11 analytes are separated in less than 40 s. Moreover, peaks in the BSI signal arising from the sample injection and EOF, enable apparent mobilities to readily be converted into electrophoretic mobilities. This leads to excellent repeatability, with analyte electrophoretic mobilities varying from 0.39 to 1.56 %RSD over eight consecutive separations. The universal detection of inorganic ions and amino acids without prior chemical modification or additives in the BGE is an advantage of refractive index detection. A disadvantage arises from modest detection limits. Here, however, we show that submicromolar detection is possible with careful thermostatting of the thin separation capillary. A series of electropherograms are used to quantify arginine concentrations from 700 nM to 500 µM, using 50 µM Li+ as an internal standard. The resulting calibration curve leads to a calculated LOD of 376 nM and a LOQ of 1.76 µM. Diagnostically relevant amino acid panels are also separated, illustrating the potential for future applications in neurodegenerative and metabolic disease diagnostics. HSCE combined with BSI detection, therefore, is shown to be a rapid, sensitive, and universal approach for analyzing sample mixtures.

Amino Acid Separation and Detection

Separation of three inorganic ions and eight amino acids in a BGE of 4 M acetic acid (pH 1.6) at a field strength of 450 V/cm. The assignment of each peak is shown in the inset, and all 11 species are separated in approximately 2 minutes at this field strength. Peaks due to the injection process and EOF are also present in the BSI signal. The shoulder on the Na+ peak is a system peak that is present in control experiments.

Rapid and Repeatable Separations

Repeated separation of the 11-component mixture at a field strength of 900 V/cm. The electropherogram in (A) shows the continuously recorded BSI signal during the 8 sample injections. (B) and (C) show expanded views of the electropherogram from the 8th injection. As discussed, the shoulder on the Na+ peak arises from a system peak that is present in control studies.

Extending BSI into the Nanomolar Regime

(A) Series of electropherograms detecting arginine as a function of concentration. The electropherograms were collected at a field strength of 900 V/cm and 50 µM Li+ was included in all samples to act as an internal standard. The inset shows an expanded view of the peak detected for the 700 nM Arg sample, illustrating the low detection limits possible. (B) Calibration plot showing excellent linearity in the 700 nM to 500 µM range measured.