Taylor Dispersion Analysis (TDA) is a microcapillary flow technique that enables solution-based sizing of small molecules, peptides and proteins, and samples with mixtures of these species. It works by measuring the time-evolved concentration profile (or Taylorgram) of a nanoliter volume sample pulse injected into a laminar flow of matched buffer. From analysis of this Taylorgram, the molecular diffusion coefficient and hence hydrodynamic radius of solute molecules can be determined.
UV absorbance is used to detect target molecules at fixed window positions along the microcapillary, with different wavelengths available to optimize measurement sensitivity and selectivity. The ability to baseline against a matched sample buffer allows label-free characterization of size and stability of biomolecules in solution, even in the presence of complex mixtures of excipients and surfactants, which are effectively rendered invisible using this technique.
Taylor Dispersion Analysis with UV area imaging detection offers mass-weighted sizing measurements that are not adversely affected by the presence of a small amount of aggregates, meaning samples can be run without dilution or filtration.
The principle of a Taylor Dispersion measurement is as follows:
- A small sample pulse of several nanoliters is injected (at t0) into a laminar flow of matched buffer in a microcapillary, under a constant drive pressure
- The sample pulse broadens as it flows along the microcapillary due to dispersion (axial direction) and diffusion (radial direction)
- UV detection at fixed windows along the microcapillary is used to analyze the absorbance at cross-sections of the sample pulse (from t1 to tn)
- Absorbance is plotted as a function of time to produce a concentration profile, or Taylorgram
- The width of the Taylorgram is related to the molecular diffusion coefficient (D) of the solute species in the sample, and hence hydrodynamic radius (Rh) can be determined
The detection of different size populations in mixtures (e.g. monomer and oligomer) is possible using Taylor Dispersion Analysis due to the use of UV detection, which provides a mass-weighted analysis. The ability to observe the contribution of a molecule using a property that is independent of size offers potential advantages in the characterization of challenging samples in formulation development. Such samples include those generated during stability studies e.g. aggregated solutions, excipient-laden formulations or those in complex biological media.