17
Robust Separation of Polar Compounds Utilising Porous Graphitic Carbon (PGC)
by Jamil Ali1 , Simon Linke2 , Luisa Pereira3 *
1GE Healthcare, Medical Diagnostics, Discovery, Amersham, UK, 2 Runcorn UK, *
luisa.pereira@
thermofisher.com - corresponding author
AstraZeneca, Research and Development, Macclesfield, UK, 3 Thermo Fisher Scientific,
The ability to retain and separate very polar, hydrophilic molecules in reversed-phase chromatography can be challenging and problematic. Retention may require the use of ion pair reagents, mobile phase pH modification, the use of concentrated buffers, or highly aqueous mobile phases. Such options have potential detrimental effects upon low-wavelength UV detection and reduced sensitivity in electrospray (ESI) mass spectrometry, and often still offer poor retention.
An example of such a challenging separation of very polar pharmaceutical analytes is shown in Figure 1. The methodology used to obtain this separation exhibited poor retention, with analytes eluting near the solvent front, and poor resolution of some components. Detection at very low wavelength was required, which limited the choice of the mobile phase and buffer. A range of columns, including polar end- capped phases, did not offer acceptable chromatography for the compounds of interest. A separation method was required for the analysis of this drug (analyte 4) in development at AstraZeneca, its degradation products, and the impurities arising from the synthetic process (analytes 1, 2, 3). Confidentiality prevents the disclosure of the name of the drug. Table 1 includes some of the physicochemical properties of these
compounds. High pKa values show that the compounds are permanently charged in conventional HPLC conditions, and the negative log D values show that the compounds are extremely hydrophilic.
Retention mechanisms of polar compounds on porous graphitic carbon
The Hypercarb material has the ability to retain very polar compounds and has other unique properties as a stationary phase in HPLC [1-4]. Its chemical surface properties distinguish PGC from more conventional LC packings such as bonded-silica gels and polymers. PGC particles are spherical and fully porous with a porosity of approximately 75%. The surface of PGC is crystalline and highly reproducible with no micro pores. At
Figure 1: The separation of 4 extremely polar compounds using a historical method on a C18 column [1].
the molecular level, PGC is made up of sheets of hexagonally arranged carbon atoms linked by the same conjugated 1.5- order bonds which are present in any large polynuclear aromatic hydrocarbon [3].
PGC behaves similarly to a strongly retentive alkyl-bonded silica gel for non-polar analytes; however its retention and selectivity behavior toward polar and structurally related compounds is very different. The retention of polar compounds can be explained by the polar retention effect (PREG – polar retention effect on graphite [3]) whereby solutes of increasing polarity showed a high affinity towards the graphite surface. With conventional alkyl-bonded silicas, the addition of a polar group to a molecule will normally reduce retention in the reversed-phase mode whereas with PGC retention is reduced to a much smaller extent
or may even increase. Tanaka and co-workers [5] plotted log k for the various stationary phases against log P (where P is the octanol- water partition ratio). For C18 the correlation was very good, but this was not the case for PGC. The retention of polar compounds on PGC was much higher than expected, exhibiting k values 4 to 15 times higher than expected on the basis of their log P. This behaviour makes PGC well suited to the separation of very polar and ionised solutes such as carbohydrates and compounds with several hydroxyl, carboxyl, amino and other polar groups [6 -11].
The retention mechanism by graphite from aqueous / organic eluents is determined by the balance of three factors [3]:
a. Hydrophobicity effect, arising from resistance to the disruption of the structure
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