Structural Elucidation

Capabilities at a Glance

  • Biotage and ISCO preparative normal and reverse phase LC
  • Forced Degradation: increase impurity level
  • Waters Preparative LC-UV-MS with mass-directed capabilities
  • NMR Experts: 1D/2D NMR data acquisition/interpretation
  • Mass Spectrometry Experts: Hi Res parent and fragment interpretation
  • Crystallization: increase the relative amount of impurity in mother liquor
  • Crystallization: suitable for single crystal X-ray analysis
  • Impurity Synthesis: scale up to produce multi gram quantity of elucidated impurity

Personnel & Experience

  • 3 NMR Spectroscopists (82 years collective experience)
  • 1 Single Crystal Diffractometer Expert (25 years experience)
  • 35 Process Chemists
    • Biotage/ISCO flash chromatography
    • Preparative TLC
    • Crystallization and synthesis
  • 10 Analytical Chemists
    • Method development (achiral/chiral)
    • Prep LC specialists
  • 7 Crystallization scientists

Illustrative Workflow

Equipment & Example Workflow

Isolation/Purification (Scope of Equipment)

We currently have a Waters ZQ/2487 mass/UV triggered auto purification LC/PDA/MS system. This system can automatically collect chromatographic peaks via UV, mass or both signal detection.   We have a variety of reverse phase columns ranging in various stationary phases and a variety of sizes from semi-preparative (10 mm diameter) to preparative scale 20 -30 mm diameter. This is useful to match the preparative chromatography with the analytical chromatography as closely as possible, or to improve currently available separation on the preparative scale, allowing for a quick and efficient isolation. We also have one normal phase chiral preparative column for chiral separation and one normal phase preparative column. This allows for efficient separation of components for purification/isolation with high chromatographic resolution.

We can purify low level components (0.1% level) typically to provide sub-milligram to milligram quantities for NMR structure elucidation. Higher level components on the percent level can, in general, be isolated more easily with greater quantities obtained, typically on the multi milligram to tens of milligram level. Main components, those present at greater than about 50%, can typically be isolated with total amounts on the 10’s to 1,000’s of milligram scale quantities.

Isolation Strategies

In terms of isolation strategies, we can also force degrade the main compound to determine if the impurity is increased. This degraded material with increased amount of impurity will make the isolation by LC-UV-MS preparatory chromatography much easier. If the impurity amount cannot be increased, we can also use any existing crystallization techniques to reject the main compound and recover mother liquors that are richer in impurities of interest for easier isolation. We can also analyze any existing mother liquor’s that may be enriched in desired impurities and use the mother liquors for impurity isolation if they contain larger amounts of desired impurities. ISCO/Biotage flash column systems can also be used to reject the main compound and recover mixtures that are enriched in desired impurities for LC-UV-MS preparatory chromatography isolation. These techniques have been employed in recent projects for isolating impurities that are at the 0.1 % level in the main compound.

NMR Data Acquisition & Interpretation

Once a suitable amount of a relatively pure isolate (greater than roughly 85%) is obtained, we can acquire 1D/2D NMR data using our Varian 500 MHz and/or Bruker 400 MHz NMR. The data sets not only encompass conventional proton-proton and proton-carbon correlation, but also proton-nitrogen correlation experiments to allow for unambiguous structural assignments.  The Varian instrument is equipped with a 3 mm inverse probe that can be used for samples that are on the sub-milligram level. We are currently in the process of acquiring a Varian cryoprobe that will allow easy data acquisition of sub-milligram quantities. The Bruker system has a multi nuclear probe that enables hetero atom 1D data collections such as 13C, 19F, 31P and more exotic nuclei such as Pt, Cl, Na, etc., as needed. It is preferable to have at least 2 milligrams for NMR analysis but meaningful data can be acquired on as little as 1/2 milligram.

Derivation of the Structural Formula

Once a complete NMR data set is obtained, a structural formula can be determined that fits the NMR data, as well as the mass spectrometry parent and fragment structures. Also, high resolution MS and fragmentation MS data can be used, if needed. A good structural formula would be consistent with all the NMR and mass spectrometry data. As an add-on bonus, we can recover the impurity from solution and provide it as an HPLC marker for analytical development.

After an unambiguous structure for the impurity is obtained, if desired, we can employ our process chemistry group to synthesize the impurity and scale up production to multigram quantity. The advantage of synthesis and analysis under one roof is that the two teams can work together seamlessly. For instance, as synthetic routes are scouted, we can analyze using LC-MS and NMR using our knowledge from the impurity isolation and identification work, allowing us to find the quick and efficient route to obtain the desired product. This will facilitate development of a quick and efficient synthetic route and speedy scale up.

Equipment

Impurity Characterization#
NMR systems (1 x 300 MHz, 2 x 400 MHz, 1 x 500 MHz)
• Varian 500 with 3 mm inverse probe for sub-milligram quantities new in 2019
New – Varian proton/fluorine cryoprobe at 500 MHz
• Bruker 400 multinuclear probe for hetero nuclei NMR (19F, 31P, 15N, etc.)
• NMR Characterization of less than 1 mg samples and mixed fractions
4
HPLC/UPLC systems
• Obtain analytical verification and purity of isolate(s)
>30
CAD units
• Obtain analytical verification and purity of isolate(s)
4
HPLC/UPLC-MS
• Use MS-MS fragmentation data to assign whole or parts of impurity structure
7
GC-MS
• Use MS-MS fragmentation data to assign whole or parts of impurity structure
3
UPLC-Orbitrap MS (accurate mass) new in 2018
• Obtain parent and fragment formula data to assign whole or parts of impurity structure
1
Single Crystal X-ray Diffractometer new in 2018
• Bruker D8 Quest
• Establish unambiguous structure and absolute stereochemistry
1

Adjunctive Solid State Support #
XRPDs
• PANalytical Empyrean Diffractometer [High Throughput] new in 2019
• Bruker D2 Phaser
• Rigaku MiniFlex
• PANalytical
4
SEM
• Nanoimages SNE-4500M with Xflash 630M light element SD detector and MCM-100 Sputter coater
1
TGA/DVS systemsmultiple
DSC systemsmultiple