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11


Pyridyl-Urea (PPU) and (iv) GreenSep Nitro (NO2


). The analytical column dimensions


were 100 x 3.0 mm i.d., 5 µm and the preparative column dimensions were 150 x 30.0 mm i.d., 5 µm. The Naphthyl and Nitro columns were supplied by ES Industries, the 1-AA by Waters and the PPU by Princeton Chromatography. The four columns were selected based on their different selectivity when tested on novel compound mixtures across different Lilly compound libraries. Figure 1 illustrates the distribution of SFC columns selected for the isolation of each target compound, from 1,000 sequential purifications across multiple chemistry projects. This exemplifies the need for a column screen, where one single column is the preferred choice for only 30% of the entire sample set.


SFC-MS methodology


The analytical and preparative SFC-MS experimental conditions are described in Figure 2. One generic analytical SFC-MS gradient method is used across all columns, with a single modifier of methanol with ammonia (20 mM). Analytical retention time windows determine the appropriate tailored purification method. See Table 1 for the five tailored gradient programs set up within the open access interface. Analytical SFC-MS screens, submitted towards the end of the day, are automatically batched by method to reduce the frequency of switch methods. Eliminating venting and pressure stabilisation steps, by minimising column switching, increases the efficiency of queued batches and helps to reduce unplanned maintenance intervention. Analytical SFC-MS screens, submitted during core hours, are batched by sample because the set of results are required more urgently to enable purification within the same day. In our experience, it is not necessary to perform scale-up calculations to account for the difference in CO2


density


and mass flow between the analytical and preparative systems. Chromatography from the preparative separation closely resembles the chromatography from the analytical separation and is generally ‘good enough’ without flow rate or backpressure adjustment.


Results and Discussion


SFC is the preferred technique for small scale, front line, achiral purifications. This assumes reasonable separation, adequate solubility in MeOH and a sample amount of less than 1.5 g. Submitted weight amount per injection, in a walk-up environment, can


Analytical Retention Time Window (min)


0.10 – 0.45 0.45 – 0.75 0.75 – 1.05 1.05 – 1.20 1.20 – 1.60


Gradient Program


1 2 3 4 5


Table 1: Modifier compositions used for the tailored preparative SFC-MS methods.


% Modifier starting conditions 5


10 15 20 25


vary considerably. Loading up to 800 mg of material is possible, but a general guideline of 50 mg per injection is advertised as an appropriate starting point. Sample quantities greater than 1.5 g require too many injections for daytime processing. Larger scale samples are directed towards flash chromatography or submitted to run by SFC overnight, using a night-time submission feature within the open access software.


One of the main challenges to the application of MS directed SFC purification in open access, is the unknown solubility of the mixture in methanol as it hits the stream of CO2


% Modifier at time 4.5 minutes 10 20 25 30 40


% Modifier at time 5.0 minutes 55 55 55 55 55


% Modifier at time 6.0 minutes 55 55 55 55 55


The standard instrument configuration introduces the CO2


stream, to the sample


flow path, directly after the injection valve, where the sample is then carried some distance to the column. If solubility is an


issue, precipitation occurs between the CO2 mixing point and the separation column. The frequency of manual intervention (to replace


post injection valve. Complex


mixtures contain many impurities with unknown solubility characteristics, as a result filters are often blocked and require intervention from an expert. Implementation of a simple solid phase extraction (SPE) sample pre-treatment step, performed by the chemist, significantly reduces (i) the risk of blockages and (ii) the overall quantity of material to process. We have also found it beneficial to make a small change in the configuration of the preparative SFC system.


filters) can be reduced by moving the CO2 mixing point to inside the column oven, just before the column switching valve. Reducing the length of tubing between the mixing point and the column, combined with heating to 55°C, helps to maintain sample dissolution and reduces the risk of sample plugging.


To mitigate risk of instrument down time, we have found it highly beneficial to support an assisted open access service where chromatographers are available to collaborate with chemists, by monitoring and assisting the purification workload. Facilitating open access purification, using a super-user, also improves efficiency by maximising instrument utilisation throughout the day and by stacking sequences to run into the evening.


Figure 1: Column type selected for SFC purification, for a set of 1,000 sequential purifications across multiple chemistry projects.


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