Carboxylate Modified Porous Graphitic Carbon: A New Class of Hydrophilic Interaction Liquid Chromatography Phases M. Farooq Wahab, Mohammed E. A. Ibrahim, and Charles A. Lucy* Department of Chemistry, University of Alberta, Gunning/Lemieux Chemistry Centre, Edmonton, Alberta T6G 2G2, Canada * S Supporting Information ABSTRACT: Stationary phases for hydrophilic interaction liquid chromatography (HILIC) are predominantly based on silica and polymer supports. We present porous graphitic carbon particles with covalently attached carboxylic acid groups (carboxylate-PGC) as a new HILIC stationary phase. PGC particles were modified by adsorbing the diazonium salt of 4-aminobenzoic acid onto the PGC, followed by reduction of the adsorbed salt with sodium borohydride. The newly developed carboxylate- PGC phase exhibits different selectivity than that of 35 HPLC columns, including bare silica, zwitterionic, amine, reversed, and unmodified PGC phases. Carboxylate-PGC is stable from pH 2.0 to 12.6, yielding reproducible retention even at pH 12.6. Characterization of the new phase is presented by X-ray photoelectron spectroscopy, thermogravimetry, zeta potentials, and elemental analysis. The chromatographic performance of carboxylate-PGC as a HILIC phase is illustrated by separations of carboxylic acids, nucleotides, phenols, and amino acids. H ydrophilic interaction liquid chromatography (HILIC) 1,2 can often retain and resolve hydrophilic analytes such as pharmaceuticals that are difficult to separate by reversed phase liquid chromatography (RPLC). In HILIC, a hydrophilic stationary phase and an organic rich (e.g., >60% ACN) aqueous eluent are used. Polar stationary phases (mainly ion exchangers) have been used with organic rich aqueous eluents for the separation of hydrophilic analytes since the early chromatographic literature. 3,4 However, it was not until 1990 that the term “HILIC” was coined by Alpert. 1 In HILIC, a stagnant water layer forms on the surface of the hydrophilic stationary phase. Analytes partition between the stagnant water rich solvent layer and the moving organic rich eluent. 1 The primary mechanism of retention is postulated to be partitioning of the analyte into this water rich layer. However, adsorption, ion-exchange, dipole-dipole interaction, hydrogen bonding, 5-7 π-π, and n-π interactions 8 also contribute to retention on HILIC stationary phases. The majority of the stationary phases employed in HILIC are based on silica, polymer, or pure inorganic oxides such as TiO 2 and ZrO 2 . Surface modification can alter the selectivity of silica and polymer HILIC phases. Neutral HILIC phases include diol, amide, and cyanopropyl. Zwitterionic phases are mainly based on sulfoalkylbetaine chemistry. Positively charged phases include those containing aminoalkyl groups. Negatively charged HILIC phases may be underivatized silica or polysuccinimide derivatized silica. 3,7 Hence, a variety of selectivities are available with silica and polymer based HILIC phases. However, silica has limited chemical and temperature stability, especially at high and low pH. This fragility is a liability for biological samples since highly acidic or alkaline washing steps (100 mM acid or base) are often required to elute any irreversibly bound matrix. 9 Polymeric particles overcame the pH stability issue associated with silica, but earlier polymer phases had limitations associated with swelling-deswelling in organic solvents. 10 More recent commercial polymeric HILIC phases such as ZIC-pHILIC, apHera-NH 2 , and Frulic-N are stable in common HILIC solvents. 7 Porous graphitic carbon (PGC) was introduced by Knox et al. 11 as a chemically robust reversed phase, an alternative to overcome the drawbacks of bonded silica phases. PGC is pH stable from 0 to 14, shows very low bleed in mass spectrometry, and can be used at temperatures up to 200 °C. 12 Some chromatographers refer to PGC as a “super reversed phase” 13 since 20-40% higher organic modifier is needed to yield the same retention as on a conventional reversed phase. Paradoxi- cally, PGC also retains highly polar analytes, such as arsenic species, 14 nucleosides, nucleotides, sugars, 15 and lipid linked oligosaccharides. 16 This polar retention effect on graphite (PREG) 17 is due to induced dipole interactions between the analyte and the graphitic surface. 18,19 In the presence of organic rich aqueous phases, the unmodified PGC shows only weak Received: January 15, 2013 Accepted: May 23, 2013 Article pubs.acs.org/ac © XXXX American Chemical Society A dx.doi.org/10.1021/ac400350x | Anal. Chem. XXXX, XXX, XXX-XXX