Nanoparticle Analysis by Online Comprehensive Two-Dimensional Liquid Chromatography combining Hydrodynamic Chromatography and Size-Exclusion Chromatography with Intermediate Sample Transformation

Abstract

Polymeric nanoparticles have become indispensable in modern society with a wide array of applications ranging from waterborne coatings to drug-carrier-delivery systems. While a large range of techniques exist to determine a multitude of properties of these particles, relating physicochemical properties of the particle to the chemical structure of the intrinsic polymers is still challenging. A novel, highly orthogonal separation system based on comprehensive two-dimensional liquid chromatography (LC × LC) has been developed. The system combines hydrodynamic chromatography (HDC) in the first-dimension to separate the particles based on their size, with ultrahigh-performance size-exclusion chromatography (SEC) in the second dimension to separate the constituting polymer molecules according to their hydrodynamic radius for each of 80 to 100 separated fractions. A chip-based mixer is incorporated to transform the sample by dissolving the separated nanoparticles from the first-dimension online in tetrahydrofuran. The polymer bands are then focused using stationary-phase-assisted modulation to enhance sensitivity, and the water from the first-dimension eluent is largely eliminated to allow interaction-free SEC. Using the developed system, the combined two-dimensional distribution of the particle-size and the molecular-size of a mixture of various polystyrene (PS) and polyacrylate (PACR) nanoparticles has been obtained within 60 min.

Nanoparticle heterogeneity: an emerging structural parameter influencing particle fate in biological media?

“…The subsequent polymer SEC analyses gave insights on the polymer Mw and Mw distribution in the different NP fractions. Larger particles generally were composed of larger polymer chains, while Mw distribution was found to be modulated according to particle size 30 . The significance of these differences in polymer composition on different biological response remains to be established.…”

“…In the case of polymeric NPs, the polymer composition and Mw can vary with NP size as demonstrated by Pirok et al The authors developed a two-dimensional liquid chromatography (LC/LC) system, which combines hydrodynamic chromatography (HC) and SEC. The HC was used to fractionate a mixture of polystyrene and polyacrylate (PS/PACR) polymeric NPs based on their size, and SEC analyzed polymers composition of each NP fraction 30 . PS and PACR NPs were effectively separated in different fractions, but the technique showed a lack of resolution to separate PACR NPs of different size.…”

Optimizing separations in online comprehensive two‐dimensional liquid chromatography

“…When used as 2 D separation, there is a wider choice of detectors and re‐equilibration is not necessary. In fact, overlapping injections allow cycle times much shorter than the 2 D analysis time .…”

“…While Table reflects the strengths and weaknesses of different combinations of retention mechanisms, challenging combinations are not useless. For example, recently a HDC × SEC separation system was developed for the characterization of polymeric nanoparticles . The authors combined an aqueous HDC separation of the particles in 1 D with an organic‐SEC separation in 2 D, using a mixer and active‐modulation traps to dissolve the particles and to switch from aqueous to organic solvents respectively.…”

Recent applications of chemometrics in one‐ and two‐dimensional chromatography

“…However, when applied to highly complex samples even with 2D chromatography, it can still be difficult to extract accurate and correct information from the obtained results. Indeed, samples such as copolymer formulations [9,10], food [11,12], protein digests [13,14], metabolic mixtures [15], and oil mixtures [16][17][18] may easily contain thousands of different components. To resolve these, powerful separation systems are needed, often equipped with sophisticated detectors such as high-resolution mass spectrometers that are able to generate huge amounts of higher-order data [19].…”