25 Table 1. Chromatographic Parameters Corresponding to the Figures.

Temperature (°C)

Figure 1 a Figure 1 b Figure 1 c Figure 1 d Figure 2

a gradient time (tG

80 80 80 80 30

Flow Rate (mL/min) tG

0.5 0.5 2.0 1.5 0.5

Change in mobile phase B indicated in parentheses. ) or the flow rate (F), the

latter approach is preferable as it would accomplish the shallower gradient without increasing the run time. Surprisingly, to our knowledge, there are very few examples in the literature of the deliberate use of this approach. What is required is the ability to increase the flow rate significantly without going beyond the usable pressure range and/or creating detrimental viscous heating issues [13-17] or band broadening due to kinetic effects [18-20]. With conventional HPLC the ability to do this is limited as we will quickly encounter any or all of these difficulties. Mutton utilised high flow rates to preserve gradient shallowness with conventional HPLC (particles sizes of 3 µm and 5 µm); however, the column length was reduced by a factor of 3 in this work and the result was a reduction of run time but with a loss of resolution [21].

It may seem that UHPLC would be a good option for this approach, as the use of smaller particles leads to lower plate heights even when working at linear velocities above the optimal [22-29] and because UHPLC systems are designed to work at higher pressures. However, in order to accomplish a significant reduction of the gradient steepness, a fairly substantial increase in flow rate would be necessary. And this, in combination with sub 2 µm particles, would generally put the pressure out of range of most UHPLC systems, unless using significantly elevated temperatures. For example, it was calculated that if working at the commonly used temperature of 30°C, with the column used in this study but with the particle size reduced to 1.7 µm, and with the flow rate increased by a factor of three, the resulting pressure would be 1560 bar, which is beyond on the range of today’s commercial UHPLC systems. Thus, we would have difficulties working at this pressure and would likely observe viscous heating issues as well. Shorter columns would offset

this to some extent; however, this could be detrimental to the separation, as mentioned above.

There are three modes of liquid chromatography that generally allow for higher optimal linear velocities and flatter van Deemter curves, without generating excessive pressures: High temperature HPLC [12,30-37], the use of columns packed with fused core particles [38-44], and monolithic columns [45-49]. When these techniques are used in combination, even flatter van Deemter curves would be expected. The use of high temperatures and flow rates have previously been evaluated for both fused-core [50-54] and monolithic columns [55-58]; and both column formats have previously been used for separation of related substances [49,59]. In this work, we evaluated a 2.7 µm fused core column at an elevated temperature of 80°C, with regards to the ability to obtain shallower gradients by increasing the flow rate, and therefore, without increasing the run time.

As mentioned above, the use of a method which offers some shape selectivity can also be quite beneficial in the separation of related substances, and this is particularly so when a portion of the molecular structure is planar (which is quite common for pharmaceuticals). It has been demonstrated in the literature that the degree of shape selectivity observed typically decreases as temperature increases [60-64]. A biphenyl phase was used in this work as it is known to offer shape selectivity [65-71], and previous experience in our laboratory (unpublished) has led us to believe that the shape selectivity offered by this phase would be less effected by temperature. Another unique feature of biphenyl columns is that they contribute both π-π and hydrogen bonding interactions which can often be helpful in the separation of molecules with aromatic rings, as in the present work.

Gradient (minutes) 26.7 Δɸa 0.8 (5 to 85%B)

106.7 0.8 (5 to 85%B) 26.7 35.6 120

0.8 (5 to 85%B) 0.8 (5 to 85%B) 0.9 (5 to 95%B)

Steepness per Relationship (1)

0.539 0.135 0.135 0.135 0.135

3. Experimental 3.1. Reagents and Materials

All chemicals used for the study were reagent grade or better. Mobile phase A consisted of 99.0% water, 0.6% acetonitrile, and 0.4% tetrahydrofuran. Mobile phase B consisted of 67% acetonitrile and 33% tetrahydrofuran. Approximately 0.1% formic acid was added to both mobile phases. The sample used for all experiments consisted of a pharmaceutical formulation that had been aged for 6 months at 40°C. The sample was prepared by weighing 2.5 g of drug product into a 5 mL flask and bringing to final volume with methanol containing 0.1% formic acid, resulting in a concentration of 25 mg/mL for the active ingredient. The structure of the active ingredient is proprietary; however its empirical formula is C16

H13 F2 N3 O3 , it has a

molecular weight of 333 g/mole, contains several rings within its structure, and a portion of the molecule is planar. An aged sample was used deliberately so as to ensure the presence of related substances. The column used in this study was a Raptor biphenyl purchased from Restek, 3x100 mm, 2.7 µm.

3.2. Instrumentation

An Agilent 1200 HPLC system was used, with a G1312A Binary Pump, a G1315B Diode Array Detector, and a G1329A Automatic Sampler (Agilent Technologies, Wilmington, DE, USA). All runs were made with a 10 µL injection and data were collected at a wavelength of 301 nm.

Several minor modifications were made, to the HPLC system, to ensure optimal results when working at elevated temperatures. First, the system was plumbed such that the mobile phase went through both sides of the heating block (whereas typically only one side is used) and an extra length of tubing, of dimensions 500 mm x 0.17 mm ID, was placed between the heating block and the column in order to ensure that the mobile phase had sufficient time to pre-heat, particularly given the fairly high flow rates that were used in these experiments. Additionally, an extra length of tubing, of dimensions 600 mm x 0.17 mm ID, was placed between the column and the detector, with approximately 100 mm of this tubing placed into a 400 mL beaker filled with water, in order to cool the mobile phase prior to reaching the detector. Lastly, a 100 psi back pressure regulator obtained from Upchurch Scientific® was placed downstream of the detector to prevent any boiling of the mobile phase.

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