Poly‐N‐hydroxyethylacrylamide as a novel, adsorbed coating for protein separation by capillary electrophoresis

Abstract

We present the polymer poly-N-hydroxyethylacrylamide (PHEA) (polyDuramide) as a novel, hydrophilic, adsorbed capillary coating for electrophoretic protein analysis. Preparation of the PHEA coating requires a simple and fast (30 min) protocol that can be easily automated in capillary electrophoresis instruments. Over the pH range of 3-8.4, the PHEA coating is shown to reduce electroosmotic flow (EOF) by about 2 orders of magnitude compared to the bare silica capillary. In a systematic comparative study, the adsorbed PHEA coating exhibited minimal interactions with both acidic and basic proteins, providing efficient protein separations with excellent reproducibility on par with a covalent polyacrylamide coating. Hydrophobic interactions between proteins and a relatively hydrophobic poly-N,N-dimethylacrylamide (PDMA) adsorbed coating, on the other hand, adversely affected separation reproducibility and efficiency. Under both acidic and basic buffer conditions, the adsorbed PHEA coating produced an EOF suppression performance comparable to that of covalent polyacrylamide coating and superior to that of adsorbed PDMA coating. The protein separation performance in PHEA-coated capillaries was retained for 275 consecutive protein separation runs at pH 8.4, and for more than 800 runs at pH 4.4. The unique and novel combination of hydrophilicity and adsorptive coating ability of PHEA makes it a suitable wall coating for automated microscale analysis of proteins by capillary array systems.

Protein attachment onto silica surfaces – a survey of molecular fundamentals, resulting effects and novel preventive strategies in CE

“…Nevertheless, basic proteins showed an increase in signal intensity and N during initial runs at pH 4.4 until an equilibrium was reached. Apparently, irreversible protein adsorption onto uncoated capillary sections had to take place or the small thickness of the polymer layer was inefficient in capillary charge shielding and protein adsorption occurred on top of the PHEA layer [218].…”

“…Apparently, polymer properties, which reduce protein binding counteract effective adhesion to SiOH [217]. Thus, efficient polymers have to meet a critical balance between hydrophilicity and hydrophobicity to allow for strong surface attachment in combination with weak protein interaction [218].…”

Electroosmotic flow suppression in capillary electrophoresis: Chemisorption of trimethoxy silane-modified polydimethylacrylamide

“…A dynamic wall coating, due to its simplicity, is a convenient way to modify capillary wall properties. Several polymers, including polydimethylacrylamide (PDMA) [9], epoxy poly(dimethylacrylamide) (EPDMA) [14][15][16], and poly(-hydroxyethylacrylamide) [10], were used to create a dynamic coating. The exposure of silica surfaces to very dilute solutions of these polymers causes development of dense polymer layers via hydrogen bonding, monopolar, dipolar, or hydrophobic forces.…”

“…An effective modification of surface properties can be achieved by coating the wall with a polymer. Neutral polymers, chemi- [4][5][6] or physisorbed [7][8][9][10] on the capillary wall, strongly decrease EOF by shielding surface charge and by increasing local viscosity.…”

Surface treatment and characterization: Perspectives to electrophoresis and lab‐on‐chips

“…Numerous other materials are able to achieve dynamic coating, such as PEO [155]. Albarghouthi et al [156] used the poly(N-hydroxyethyl acrylamide) (PHEA), which was synthesized by free-radical polymerization. The hydrogen interaction of the hydroxyl groups with the capillary surface increases the coating stability.…”