18
May/June 2013
of hydrogen-bonded solvents by non- hydrogen-bonding analytes. This is primarily a solution effect that tends to drive analytes out of solution.
b. Dispersive interactions of the London type between the graphite surface and the analytes. These are largely balanced by similar interactions between the graphite surface and the eluent (a) which is displaced by the analyte. Their net effect may either encourage or discourage retention, but they may have an important effect on selectivity.
c. Interaction of polarisable or polarised groups in the analyte with the polarisable surface of the graphite (Figure 2). These are additional to the normal dispersive interactions.
The overall effect of these competing interactions is that increasing the hydrophobicity of the analyte, for instance by adding alkyl groups into a molecule, always increases retention, as expected in a typical reversed-phase mode. However, increasing the polarity of the analyte by adding groups which can either donate or accept electrons or can polarise the graphite surface may also increase retention, particularly if these groups are constrained to be in close contact with the graphite surface. Therefore, the strength of interaction depends on both the molecular area of an analyte (and, therefore, shape of the analyte) in contact with the graphite surface and upon the nature and type of functional groups at the point of interaction with the flat graphite surface. The flatter the analyte, the closer its alignment to the graphite surface with a higher number of points of interaction possible, the greater the retention.
Goal
The purpose of the work described herein was to develop a methodology that can be effectively utilised for the retention of extremely polar compounds such as those described in Table 1. Porous graphitic carbon columns (Hypercarb) were found to provide suitable retention and separation of the analytes under analysis. The optimised method uses conventional LC mobile phase conditions that allow UV detection at a low wavelength of 195nm. Two versions of the method are described. The first is suitable for systems with low dwell volume (less than 100 µL), such as UHPLC equipment. This combination allows the use of narrow bore
Figure 2: Schematic representation of charge induced interaction at the PGC surface.
columns and fast gradients, resulting in 3× faster methods using approximately 11x less solvent. The second method was developed for systems with higher dwell volume (approximately 1100µL) and higher dead volume (such as those found in conventional HPLC equipment). This meant that a 4.6mm diameter column was essential to maintain an appropriate ratio between column volume and extra-column volume to minimise associated band-broadening. The second method also has two additional compounds (by-product 5 and component 6) added, which were incorporated as methodologies for different stages of manufacture were combined.
Experimental Method development background
The application described herein entered development at AstraZeneca using a C18 column, whereby polar components were eluted isocratically under the starting conditions of 100mM potassium phosphate / acetonitrile (95:5, v/v) mobile phase [12]. A gradient section was present extending to 50% acetonitrile to ensure the elution of any late eluting impurities. Analysis time was 60 minutes per injection and exhibited poor retention of the newly identified polar components (analytes 2 and 3), as shown in Figure 1. Typically within AstraZeneca, new methods are developed through a process of screening a small range of orthogonal stationary phases, organic solvents and pH conditions. This identifies a suitable method in the majority of cases, often with little to no optimisation. Polar species are one area where this standardised approach to method development can fail to deliver a suitable method, as was the case in this application
where no suitable methodology was identified under any of the conditions.
The separation in this application was hindered by the extent of the limitations presented by the analytes of interest. Poor UV chromophores necessitated detection at 195nm, reducing the number of available mobile phase options based on UV cut-off. Poor solution stability limited the range and concentration of pH modifiers that could be employed and poor solubility in non- aqueous solvents further limited the separation options.
Retention mechanisms involving HILIC, mixed-mode or normal phase approaches are typically used for the separation of polar species; these were all assessed but within the above restrictions could not be used to produce a suitably robust method.
The simplest next step approach was chosen: to assess the separation in 100% aqueous conditions. However, traditional C18 columns
Compound 1 2 3 4 5 6
Calculated pka
pka (1) >14 pka (2) 7.4
pKa (1) >14 pKa (2) 1.7
pKa (1) >14 pKa (2) 1.8
>14 Neutral >14
Calculated LogD (at pH 4.5)
-3.7 -7.0 -7.1 -5.4 n/a 0.6 Table 1: Physicochemical properties of the test compounds
(calculated values obtained using ACDLabs pKadB 12.01 software).
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