The full name of LC-MS is liquid chromatography-mass spectrometry.
MS is an instrument capable of generating ions. Therefore, LC-MS is an instrument that separates and detects ions in the gaseous state according to their mass-to-charge ratio.
LC/MS uses liquid chromatography as the separation system and mass spectrometry as the detection system and thus has the characteristics of both high separation in liquid chromatography and high sensitivity in mass spectrometry.
The HPLC-MS sample is separated from the mobile phase in the mass spectrometry section, is ionized, and then the ion fragments are separated by mass number by the mass analyzer of the mass spectra, which is obtained by the detector. LC-MS reflects the advantage of complementary chromatographic and mass methods, combining the high separation ability of chromatographic method for complicated samples with the high selectivity advantages of mass spectrometry, highly sensitive and providing relative relative molecular mass and structure information, it has been widely used in many areas such as pharmaceutical analysis, as well as food analysis and environmental analysis.
The working principle of LC-MS is as follows: After the sample is separated by liquid chromatography, each component enters the mass spectrometer detector in turn, and each component is ionized at the ion source, producing ions with a certain charge and different mass numbers. The ions are ionized at the ion source to produce ions with different charges and mass numbers. The ions are separated by the mass analyzer according to different mass-to-charge ratios (m/z), and the mass spectra are obtained in the order of mass-to-charge ratios. The qualitative and quantitative results of the sample can be obtained by analytical processing of the mass spectra.
Liquid chromatography-mass spectrometry analysis mainly consists of HPLC (high performance liquid chromatography) system and MS (mass spectrometry) systems.
HPLC system allows for efficient separation of mixtures, volatilization of the mobile phase to produce large amounts of gas, atmospheric inlet pressure, no mass limitation, and the ability to use buffered salt solutions.
MS systems, on the other hand, require high vacuum conditions to operate, and MS systems are equivalent to mass analysis systems and are best operated with volatile buffers.
In LC-MS equipment, the HPLC system and the MS system play a crucial role in obtaining a complete mass spectrum. Firstly, the HPLC system starts the ion source system through the LC interface to focus the ion beam, and then the ion beam is focused and the sample is analyzed through the MZ analyzer and then transferred to the detector.
In addition to the ability to analyze strongly polar, difficult to volatilize, and thermally unstable compounds that cannot be analyzed by gas chromatography-mass spectrometry (GC-MS), LC-MS also has the following advantages.
a. Wide analytical range, MS can detect almost all compounds, which can solve the problem of analyzing thermally unstable compounds relatively easily.
b. Strong separation ability, even if the analyzed mixture is not completely separated on the chromatogram, but the characteristic ion mass chromatogram of MS can give their respective chromatograms for qualitative and quantitative purposes.
c. The qualitative analysis results are reliable, and the molecular weight and rich structural information of each component can be given simultaneously.
d. The detection limit is low, the MS has high sensitivity, and the detection capability can be increased by more than one order of magnitude by selected ion (SIM) detection.
e. Fast analysis time, the liquid chromatographic column used in LC-MS is a narrow diameter column, which shortens the analysis time and improves the separation effect.
f. A high degree of automation, LC-MS is highly automated.
There are two major classification systems for liquid chromatography-mass spectrometry (LC-MS) techniques in common use today, one from the perspective of the ion source of the mass spectrometer and the other from the perspective of the mass analyzer of the mass spectrometer.
| Classification perspective | LC-MS Name |
|---|---|
| Ion source | Electrospray ionization sources (ESI) Atmospheric pressure chemical ionization sources (APCI) Atmospheric pressure photoionization source (APPI) Matrix Assisted Laser Desorption Ionization (MALDI) |
| Mass analyzer | Quadrupole liquid chromatography-mass spectrometer Ion trap liquid chromatography-mass spectrometer Time-of-Flight Liquid Chromatography-Mass Spectrometer (TOF) Fourier transform mass spectrometry |
Among them, ESI, APCI, and APPI ion sources are mostly used in combination with quadrupole and ion trap mass spectrometry, which are the most widely used liquid mass spectrometers at present.
From the ion source point of view, ESI is suitable for compounds of medium to high polarity, especially for reversed-phase liquid chromatography coupled with mass spectrometry, and is currently one of the most widely used ionization methods in liquid-mass spectrometry; due to the development of pneumatic-assisted spray, the tolerable liquid-phase flow rate is increased to 1 mL-min-1; by forming multi-charge ions, the molecular weight analysis range can be extended ESI also has the advantage that it is a concentration-based detector and can therefore be used regardless of the sample volume, and the micro spray (μESI) and nanospray (nESI) techniques developed in recent years are particularly suitable for highly sensitive analysis of trace samples. APCI is a technique for ionizing analytes in the gas phase using photochemical action at atmospheric pressure, which is similar in scope to APCI and is complementary to it. MALDI has the advantages of easy coupling with TOF for the determination of high mass number molecules, high sensitivity, and simple sample preparation. MALDI has been widely used for the analysis of proteins, peptides, nucleotides, polysaccharides, and synthetic polymers. However, due to the characteristics of MALDI, there are relatively few studies on the application of direct online coupling with LC.
From the mass analyzer point of view, the quadrupole is used to make certain qualified ions pass through the quadrupole to the detector under the action of an alternating electric field; the ion trap is used to gather ions inside the trap first, and then release the ions inside the trap to the detector one by one by changing the electric parameters. Single-stage quadrupole mass spectrometry is used only for primary mass spectrometry, while three-stage quadrupole mass spectrometry can perform secondary mass spectrometry functions. The primary mass spectrometry gives molecular weight information of the compound, while the secondary mass spectrometry gives structural information such as fragment ions of the compound through collision-induced dissociation (CID). The ion trap mass spectrometry has a multi-stage mass spectrometry function (generally can achieve 5-11 levels), especially with an automatic multi-stage (Auto MS(n)) function, which is more favorable for resolving the structure of compounds and reduces the requirements for the resolution of the spectrum, but the mass accuracy and resolution are not as good as the quadrupole mass analyzer; the three-stage quadrupole can also meet the general structure resolution function, but is limited by The three-stage quadrupole can also meet the general structure analysis function, but is limited by the scanning time and is not suitable for scanning-type analysis in a large mass range. For primary mass spectrometry with selected ion detection (SIM) or tandem mass spectrometry with multiple reaction monitoring (MRM), quadrupole mass analyzers are generally 1-2 orders of magnitude more sensitive than ion traps and are therefore more suitable for quantitative analysis of trace or trace components. Time-of-flight mass spectrometry is the application of different m/z ion flight speeds, ion flight through the same path to reach the detector at different times and obtain a mass separation, it is often used in conjunction with MALDI, the advantages are fast scanning speed, analysis of a wide range of mass; Fourier transform mass spectrometry is a new technology developed in the last decade or so, its working principle and the above-mentioned mass analyzers are fundamentally different, the technique applies Fast Fourier transform method converts the frequency signal of ions into mass spectrometry signal, which has the advantage of high resolution, and the sensitivity increases with the resolution.
Electrospray (ESI): The ionization process involves the generation of charged droplets by an electric field, followed by ion evaporation to produce the ions to be analyzed. The required compound does not need to be volatile and is the method of choice for the analysis of thermally unstable compounds. The ions are already generated in solution and can generate multi-charged ions in addition to single-charged ions.
Atmospheric pressure chemical ionization (APCI): a gaseous chemical ionization (CI) process in which the solvent or reaction gas is first charged by the action of a corona needle and then transferred to the compound to form ions. In the use of APCI, the chemical and the compound need to be volatile, the chemical and the compound must be thermally stable, and the ions are generated in gaseous conditions, but only singly charged ions are generated.
Since different ion sources have different ionization modes, they have different ranges of applicability. Before performing analytical experiments, a suitable ion source should be selected according to the type of the target compound.
| LC-MS ionization mode | Scope of application |
|---|---|
| ESI+ | Suitable for basic compounds, or compounds containing heteroatoms, etc, Compounds containing group such as -NH2, -N, -NH, -CO, -COOR, etc. |
| ESI- | Suitable for acidic compounds, or compounds containing strong negative groups. Compounds containing groups such as -COOR, -OH, etc. |
In LC-MS, the most commonly used solvents for the mobile phase are methanol, acetonitrile and water, other solvents can also be used.
| Solvents for ES and APCI sources | Solutions suitable for APCI sources |
|---|---|
| Methanol Acetonitrile Water Ethanol Propanol Isopropanol Butanol Acetone | Hydrocarbons (e.g., n-hexyl firing) Cyclo-fired hydrocarbons (e.g., cyclohexane-fired) Toluene Benzene Styrene Carbon tetra-gasification Carbon disulfide |
The mobile phase additives used on LC-MS are different from LC, where only volatile additives can be used, and the common types of additives are as follows.
Positive mode
M - NH2 + Acid [M - NH3]* + Acid
Volatile acids are commonly used for selecting mobile phases containing volatile acids in positive mode: (formic acid, acetic acid)
Negative mode
M- COOH + Base [MCOO] + Base
Select the mobile phase containing volatile bases or volatile salts in negative mode commonly used volatile bases or salts < hydrogen water, formic acid press, ammonium acetate
When performing LC-MS experiments, it is important to select the correct column size according to the type of ion source and set the appropriate LC flow rate so that the proper peak shape, as well as the desired ionization efficiency, can be obtained.
