Reaction Analysis (LCMS)

When a chemist synthesizes a molecule, they need to check that the reaction actually passed, and ideally know how well it went:

  • Reactants don’t necessarily react or react in an unexpected way

  • Even if they react, the reaction may be slow and inefficient and doesn’t necessarily produce enough product

  • The reactants and/or the products may degrade and produce side products

For this reason, synthetic chemists need to run a Quality Control (QC), and using HPLC/LCMS is a common technique in this case. Typical setup:

  • Single Quadrupole Mass Spec (SQD) which provides a low m/z resolution. More precise instruments are usually unnecessary - too much complexity for a simple task.

  • Soft ionization: Electrospray (ES), Atmospheric Pressure (AP), Chemical Ionization (CI). Soft ionization allows simple peak detection and integration. While hard ionization would break the molecule - which is more suitable when we work with unknown compounds that we need to identify.

  • DAD/PDA to detect non-ionizable species and for quantification (if needed)

All the chemist needs is to specify either the molecular mass of the Analyte, or better - its chemical representation: Molecular Formula or Structure in MOL/SMILES/InChI format.

Identifying the analytes with Mass Spec

Mass Spec is used to identify if it’s our specific analyte. We know which molecules we’re looking for because we know which reactants we added and which product we expect to find.

In case of some unknown peaks, it may be hard to identify what that analyte is. In complicated situations the identification may need high-resolution mass spectrometry with Electron Ionization.

Orthogonal peak identification via DAD

DAD/UV can potentially identify molecules that can be missed by Mass Spec (if they aren’t ionized well), and at least give a hint that we should look for something else too. But without spectral libraries to match against, this won’t tell us much about the structure of the compound.

DAD/UV is "orthogonal" to Mass Spec because it uses completely different molecular properties and therefore provides a very different perspective on the analyte. "Orthogonal" as in "orthogonal vectors" that point in different directions - something that’s often used in math to decompose a complicated signal into independent components.

Quantification

Usually we can only tell if the compound is present or not, maybe guess if there’s a lot of it or not. But both Mass Spec and UV detection aren’t "universal", meaning that different compounds produce different intensities even if they have same concentration. So CompoundA can produce a big peak at low concentration, while CompoundB could be barely detectible at high concentrations.

If we want to get the actual concentrations, we first need to inject the analytes with known concentrations (we need to prepare clean standards first). DAD allows to have a single-point calibration because:

  • If DAD is properly calibrated, 0 concentration gives 0 intensity. This gives us a 2nd point to build the calibration curve.

  • Mass Spec on the other hand if often noisier and more fragile, with lower linear dynamic range

Peaksel capabilities

Reaction QC is the most popular of the supported workflows. Relevant functionality:

See also