Purification Synthetic Chemistry Sample The effort required for successful purification of synthetic mixtures with high recoveries depends on several factors. These factors include:
- • Initial purity. Cleaning up a 10% mixture is much more difficult than a 50% mixture.
- • Similarity of the desired product to the impurities. Is the needed selectivity readily achievable?
- • Solubility in chromatography compatible solvents.
- • Stability during concentration.
- • Scale. How large is the sample?
Years of experience supporting medicinal and combinatorial chemistry groups has shown us that one size methods do not work. The 80/20 rule is not sufficient. Most medicinal chemists define success as 99/1. We must be successful or promptly return the sample with a rationale of why HPLC purification will not work. Our pricing scale assumes that for low numbers of samples, every sample will be unique and require bespoke methods. As the number of samples grows, the samples become more uniform and common methods emerge. Our service is defined by looking at structures and choosing conditions to maximize the possibility of success. This is vastly different than a one-size-fits-all method. One-size methods reduce cost, but will result in a higher failure rate. Our process requires more labor but almost always results in successful purifications. Sample information:
An Excel spreadsheet (paper is OK for <10 samples) will be provided with each batch of samples. Click here for example submission form. The submission form should contain the following information:
- • Sample ID number
- • Recommended reversed phase compatible solvents (or solvents to avoid)
- • Dissolution recommendations or precautions (Does the addition of acid increase solubility or degrade the compound?)
- • The structure. In lieu of the structure we can make do with the following information: o Number of basic nitrogens o Number of acid groups o clogP
- • Any known stability issues (Does it form the methyl ester in acidified methanol?)
- • Who to contact if there are questions about the sample.
Sample format:
Medicinal chemistry samples are usually received as dry powders in vials (e.g. 4 dram). Combinatorial samples are usually received as DMSO stocks in 96 well plates. Analytical analysis:
Samples are dissolved (100 to 200 mg/mL). A small aliquot of the concentrated sample is diluted and an analytical LC-DAD-ELSD-CLND-MS analysis is performed. The purity and quantity of the desired compound is estimated. Preparative conditions are determined. Purification:
Samples are purified by reversed phase HPLC. Typically, 100mg of material is injected on a 2cm diameter C18 column. Elution is achieved by a water to methanol gradient where both solvents are acidified. For high number low mass samples (e.g. combichem), mass directed purification is used. For higher mass and lower number samples, the purification is usually UV directed. Dry down:
Fractions are pooled and concentrated by nitrogen blow down or vacuum centrifuge as appropriate. The sample is redissolved in a volatile organic solvent and transferred to a tarred vial. A small aliquot is taken for final LC-DAD-ELSD-CLND-MS analysis. The vials are evaporated and weighed. Results:
An analytical report containing the initial and final analytical data along with the final amount and purity will be provided. The vials will be packaged and shipped. Isolation Services OpAns performs UV or MS based compound isolation services. Samples are typically from natural products, in-vitro metabolism, or stability studies. Target analytes are isolated for further structural characterization (e.g. NMR) or activity confirmation. |
