Separation and Mass Spectrometry – MACAL

The Agilent InfinityLab LC/MSD is a liquid chromatograph (LC), coupled to a diode array detector followed by a mass spectrometer (MS).

LC can separate compounds on a liquid chromatography column where the coated particles inside the column act as stationary phase and a mixture of water and a solvent (typically methanol or acetonitrile) as the mobile phase. Retention times (i.e. the time the compounds take to pass through the column) on common reversed phase columns depend mostly on the compound’s polarity.

In order to be detectable by this instrument, the compounds need to absorb light in the UV/Vis wavelength range and/or be ionizable with electrospray ionization or atmospheric pressure chemical ionization.

The diode array detector is somewhat compound-specific as different compounds have different absorption maxima (i.e. the wavelength where they absorb strongest) but those maxima are fairly broad and the compounds may still absorb at many other wavelengths, just less strongly. Therefore, the LC method should separate the analytes from interfering chemicals. The diode array detector can also record a whole spectrum. For quantification, a standard of known concentration is required, unless the molar extinction coefficient is available. In this case it is possible to relate the signal intensity directly to a concentration without comparison to a standard.

The MS coupled to this LC is a low-resolution instrument, i.e. it can distinguish ions that differ by at least 1 Da. It is equipped with a single quadrupole as mass analyzer and the ionization is facilitated by an electrospray ionization source (ESI) or an atmospheric pressure chemical ionization source (APCI). Compared to electron ionization as it is often used with GC-MS, these ionization techniques are not universal - not all chemicals can be ionized and detected.

ESI is especially suited to ionize relatively polar chemicals with ionizable functional groups. The ionization happens mostly in the liquid phase. Often, compounds get protonated (positive mode) or deprotonated (negative mode), but formation of adducts with other cat- or anions is also common. ESI requires the use of polar solvents that evaporate efficiently.

APCI can also ionize some more non-polar chemicals as ionization happens in the gas phase. In theory, this allows also for using non-polar solvents such as dichloromethane or chloroform. However, such solvents cannot be used when APCI is used in combination with reversed phase LC.

Both ESI and APCI are soft ionization techniques, causing no or little fragmentation. As a result, many compounds are detected as the ionized version of the whole molecule. It is therefore not possible to identify compounds using spectral libraries.

The ionization efficiency in ESI and APCI can vary largely between chemicals. Therefore, it is not possible to infer a concentration/amount directly from the signal intensity. Standards of known concentration are crucial for quantification.

Capabilities/Accessories

The LC is currently equipped with the following columns:

• YMC-Triart C18, dimensions: 150 mm x 4.6 mm, particle size: 5 µm
• YMC-Pack C8, dimensions: 150 mm x 4.6 mm, particle size: 5 µm
• Poroshell 120 EC-C18, dimensions: 100 mm x 4.6 mm, particle size: 4 µm

Your own reversed phase column can also be installed.

Aim for a concentration of ca. 1 µM

Suitable sample solvents include methanol, acetonitrile and water. However, if your compound does not easily dissolve in those solvents you can try making a stock solution in another solvent, e.g. ethyl acetate, chloroform, DMSO, then dilute this with methanol if possible. You can inject small amounts of those solvents, but your sample must not precipitate when being diluted by the mobile phase.

The Shimadzu GCMS-QP2020 is a gas chromatograph (GC), coupled to two detectors - a mass spectrometer (MS) and a flame ionization detector (FID). If both detectors are to be used then duplicates of the samples are injected at the same time into two injectors and are separated on two separate columns of the same type.

GC can separate compounds on a capillary column where the coating on the inside of the column acts as stationary phase and helium as the mobile phase. In order to be analyzable by GC, the compounds need to evaporate at a temperature that does not cause their thermal decomposition in an inert atmosphere. The column can also only be heated to a specific maximum temperature. Retention times (i.e. the time the compounds take to pass through the column) depend mostly on the compound’s vapor pressure.

The FID is a fairly general detector for organic compounds. It is unspecific, i.e. it does not allow for distinguishing between different compounds. The signal intensity is proportional to the mass of carbon eluting at any given point in time. Therefore, if interference from coeluting chemicals is negligible, the FID can to some extent be used to estimate the absolute concentration of a known chemical without running a standard with known concentration.

The MS coupled to this GC is a low-resolution instrument, i.e. it can distinguish ions that differ by at least 1 Da. It’s equipped with a single quadrupole as mass analyzer and the ionization is facilitated by an electron ionization source (EI). EI can ionize most chemicals and the signal is compound-specific as the MS measures the mass of compounds and their fragments which are formed in the EI source. Fragmentation can be weak or very strong, depending on the chemical, i.e. for some chemicals the molecular ion will be dominating while others fragment intensely and form a few or many different fragments, depending on the compound. As the energy used for ionization is standardized at 70 eV, the fragmentation pattern is reliable and the results can be compared to large libraries in order to identify compounds. In theory, the sum of all ions formed from a chemical is proportional to the molar amount. However, in practice, the response for compounds with different functional groups may vary and comparison to standards of similar chemicals with known concentrations is necessary to relate the signal intensities to absolute concentrations. As a result, FID and MS can provide complementary data.

Capabilities/Accessories

• The GC is equipped with two HP-5MS columns, 30 m length, 0.25 mm inner diameter, 0.25 µm film thickness.
• The maximum temperature those columns tolerate is 325 °
• We have a general 16 min oven program, but individual methods can be used too. The split/splitless injector allows for injection of 1 uL of particle-free sample.

Aim for a concentration 1–10 µg/mL

Suitable solvents include hexane, dichloromethane, ethyl acetate, isooctane, acetone, toluene, chloroform etc. Methanol and acetonitrile can be used but do expand more (especially methanol), so they can easily cause spillage of sample vapor from the injector, resulting in a lower signal. Unsuitable solvents are water, DMSO, DMF etc. as they damage the column.