Liquid chromatography-mass spectrometry (LC-MS) is an instrument that combines a liquid chromatograph with a mass spectrometer for qualitative and quantitative analysis of samples. It is characterized by a modern analytical technique that combines liquid chromatography with a wide range of applications and mass spectrometry that is sensitive, specific, and can provide molecular weight and structural information. Its working principle is: after the sample is separated by liquid chromatography, each component enters the mass spectrometer in turn, and each component is ionized in the ion source to produce ions with a certain charge and different mass numbers.
Different ions have different motion behaviors in the electromagnetic field. The mass analyzer is used to separate the ions according to different mass-to-charge ratios (m/z), and the mass spectra arranged in the order of mass-to-charge ratios are obtained. Through the analysis and processing of the mass spectrum, the qualitative and quantitative results of the sample can be obtained.
LC-MS mainly includes liquid chromatography system, interface, ion source, mass analyzer, detector, vacuum system, electrical system and data processing, etc.
The liquid chromatography part is basically the same as the ordinary LC, and consists of a sampling system, an infusion system, a separation system, a detection system, etc., while in the LC-MS system, the MS part is used as the detector of the LC.
In the early days, diaphragms and stopped-flow inlets were used, which were installed at the inlet of the column. Most of them now use six-way injection valves or autosamplers. The sampling device requires good sealing, small volume, and good repeatability to ensure central sampling and little impact on the pressure and flow of the chromatographic system during sampling.
The infusion system mainly includes a liquid reservoir—used to store the mobile phase; an infusion pump—the output pressure of the high-pressure pump is generally 150-500 kg/cm2. (1 kg/cm2=98.0665 kPa), the flow rate is 0.01~10 mL/min, the requirement for the high pressure pump is constant flow rate, no pulsation, and the flow rate can be adjusted; Filter—used to filter tiny impurities; degassing device—if the air contained in the mobile phase is not removed, the air bubbles in the mobile phase will be compressed by pressure when it passes through the chromatographic column, and will be compressed when it flows out of the chromatographic column to the detector. Air bubbles are released under normal pressure, which increases the noise of the detector, makes the baseline unstable, and the instrument cannot work normally; Gradient elution device – there are two ways: one is called low-pressure gradient, which means that the solvent is mixed in a certain proportion under normal pressure and then input into the chromatographic column by a high-pressure pump, also known as external gradient; the other is called high-pressure gradient, which means that the solvent is mixed with a high pressure A pump feeds the individual solvents into a mixer where they mix and then feed the column, also known as an internal gradient.
The separation system includes chromatographic columns, connecting pipes and thermostats, etc. Compared with LC, LC-MS generally has a lower flow rate (high flow rate may affect the atomization effect), so narrow-diameter columns are generally used, that is, 2mm or 2.1mm, and the particle size is generally 3.5μm, and there are also 5μm columns. If you use an inner diameter of 4.6mm, the flow rate is the same as that of ordinary liquid phase, but it can be split after the column, so that the amount entering the mass spectrometer will not be too large.
The interface technology of LC-MS is more difficult than that of GC-MS, because the mobile phase in liquid chromatography is liquid, while mass spectrometry detects gas ions, so the interface technology must solve the problem of liquid ionization. The most commonly used ionization sources in LC-MS are atmospheric pressure electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), both of which belong to atmospheric pressure ionization (API) technology, and the ionization process takes place under atmospheric pressure.
The working principle of ESI is to turn liquid droplets into steam, and the solvent formed in the process of ion emission is transported to ESI Probe by the liquid phase pump, and flows out through the stainless steel capillary inside it. At this time, a high pressure of 2-4kv is added to the capillary. With the effect of atomizing gas, when the mobile phase flows out from the top of the capillary, a fan-shaped spray will be formed, so that the droplets generate an aerosol containing samples and solvent ions.
Electrospray ionization can be divided into three processes: 1) Formation of charged droplets: due to the high pressure applied to the capillary, redox reactions are caused to form charged droplets. 2) Solvent evaporation and small droplet fragmentation: solvent evaporates, ions move to the surface of the droplet, and the ion density on the surface of the droplet becomes larger and larger. When it reaches the Rayleigh (Rayleigh) limit, that is, when the Coulomb repulsion force generated by the charge on the surface of the droplet is roughly equal to the tension on the surface of the droplet, the droplet will break up non-uniformly and split into smaller droplets. After the mass and charge are redistributed, the smaller droplets will enter the stable state, and then repeat the series of processes of evaporation, charge excess and droplet splitting. 3) Formation of gas-phase ions: For droplets with a radius <10nm, the electric field formed on the surface of the droplet is strong enough, and the repulsion of the charge eventually causes some ions to evaporate from the surface of the droplet instead of the splitting of the droplet, and the final sample is single-charged or multiply charged ions from the solution to the gas phase, forming gas phase ions. ESI is the softest form of ionization and usually produces only molecular ion peaks. It is suitable for thermally unstable polar molecules, and can analyze small molecules and macromolecules.