Liquid protocol. The liquid protocol was aimed at analyzing solution concentrations ranging between 20 mg/mL (50 volumes) and 200 mg/mL (5
volumes); these concentrations are typical of those used in reactions. Because the 8-minute HPLC gradient method requires
sample concentrations ranging between 0.003 and 0.35 mg/mL, accurate dilutions of approximately 1000-fold were needed. Previous
experience had shown that electronic displacement pipettes (EDPs) were accurate and easy to use. To achieve a 1000-fold dilution
and retain a suitable sample volume, the developed protocol involved adding 20 microliters of solution to 20 mL of solvent
in a scintillation vial. Analytical solution concentrations are quite high, thereby requiring a low injection volume (1 µl).
The low injection volume facilitates protocols that do not rely on significant serial dilution, have minimal peak splitting
due to solvent mismatch, and do not significantly compromise the repeatability. Although acceptable accuracy and reproducibility
were demonstrated when developing a protocol for most of the commonly used solvents (e.g., toluene), certain solvents such
as dichloromethane could not be dispensed with sufficient accuracy. Because dichloromethane is a commonly used solvent, a
modification to the same protocol was made by replacing the EDP pipette with a 25-microliter syringe. A Pressmatic (Bibby
Scientific, Staffordshire, UK) dispenser was used to deliver the 20 mL of solvent (typically methanol or acetonitrile) to
the scintillation vial (this device can be attached directly on top of a bottle of solvent and is sufficiently accurate and
precise when delivering the solvent). Feedback from users also indicated that there was a need to analyze compounds across
a wider range of concentrations to support the analysis of more concentrated reaction mixtures and diluted solutions typical
of mother liquors or wash solutions to optimize yields. These protocols used the same principles to ensure that samples could
be easily prepared (see Figure 2).
Figure 2: Screenshot showing the Yieldaliser (GSK) software's assay interface.
Solid protocol. The requirements for establishing a solid protocol were similar to those used to create the liquid protocol. The developers
initially used four-decimal-place balances in the laboratories, Pressmatic dispensers, and EDP pipettes. The final protocol
calls for ~15 mg of solid that is weighed accurately on a four- or five-decimal balance into a disposable polypropylene container.
Fifteen miligrams was found to be the minimum weight required for achieving an accurate measurement. One mililiter of dimethyl
sulfoxide (DMSO) was added to the solid with an EDP pipette, and then 40 mL of solvent was added from the Pressmatic dispenser.
DMSO was chosen as the predissolution solvent based on its aproticity, which maximizes solubility while remaining compatible
with the system.
Adding and selecting compounds. A similar protocol to the solid protocol can be used to assess the suitability of a compound for the Yieldaliser and to ensure
accuracy in determining the response factor. A full assessment of safety, stability, repeatability, and applicability of the
compound (e.g., presence of appropriate λOpt ) should be performed. Using five-decimal-place balances and volumetric glassware
to determine response factors provides improved accuracy over the liquid and solid protocols described above with a percentage
relative standard deviation of < 1.5% (n = 3). Accurate determination of the response factors is crucial for meeting customer
expectations of the system's performance.
The left side of Figure 2 shows the compounds (stored under each project) for which the Yieldaliser is set to analyze. The
right side displays the custom choices a user can make when performing yield in solution- or solid-assay analysis. Figure
2 shows the screen that appears after selecting the assay option.
Figure 3: A proof-of-concept study demonstrated chemists can generate data with acceptable precision and accuracy.
Proof of concept. A proof-of-concept study was performed to demonstrate that chemists can produce data within ± 3% of the actual purity (chemists'
expectations) for the previously defined protocols. Four trained chemists were able to generate yield in solution and assay
results for samples of known concentration within these limits for several compounds. Figure 3 provides some of the data generated
from the proof-of-concept study. More than 90% of the data were within the desired ± 3% limits. The clusters of results that
fell outside the limits represent results from chemists who were not familiar with the sample preparation devices. After receiving
additional training, the data from these chemists improved in precision and accuracy.