Liquid Chromatography-Mass Spectrometry (LC-MS) is a powerful analytical technique combining the capabilities of physical separation obtained using liquid chromatography coupled to analysis of mass of chemical compounds using mass spectrometry.
It is probably one of the most common technologies available
Liquid chromatography is usually defined as HPLC and has been employed successfully for separating a range of compounds. It is one of the most powerful separation methods available especially for food and biomolecules. The addition of mass spectrometry allows for ramped up detection power.
In an LC-MS system, the detector is fitted to part of the system that interfaces with the mass spectrometer. The interface is nowadays an ionization source.
Typical Applications:
- pharmaceutical analysis – rapid identification of drugs, of metabolites and of proteins.
- biochemical applications: rapid protein identification at all levels
- clinical applications – rapid identification of drugs and of metabolites.
LC or more appropriately HPLC, is a liquid phase operation which operates as a temperature between 21 and 50°C. Thee are no limitations on mass other than the solutes must be completely soluble. The eluent flow is usually about 1ml/min for optimal resolution and which is equivalent to 500ml/min of gas if a carrier gas was used as in gas chromatography. Inorganic buffers are always preferred.
In a mass spectrometry system, operation relies on a vacuum and the temperature must be raised to be tween 200 and 300°C. It requires the use of volatile solvents and buffers
The Mobile Phase
The mobile phase is the solvent that moves solutes through the column. Mobile phases must have the following characteristics:
- low cost, high purity
- UV transparency
- low viscosity
- non-flammable
- non-corrosive especially in the LC system
- non-toxic
The solvent strength and selectivity is critical as this establishes how well the solutes are stripped from the column.
Sample Preparation
Samples for analysis are prepared by concentrating the analytes to begin with and removing any compounds that could interfere. Given the type of analysers used, any compounds which contribute to a background ionization or suppress ionization are removed.
Typical concentration methods might be to use concentration on the column itself as in HPLC, to desalt the solution so that sodium and potassium ions are removed. Diafiltration is often employed to separate low molecular weight compounds from higher molecular weight compounds like proteins and large carbohydrates.
The Interface And Methods Of Ionization In The Mass Spectrometer.
There are considerable difficulties in interfacing a liquid chromatography set-up with that of a mass spectrometer. The biggest issue is the removal of the solvent used in the LC part.
Most popular interfaces are:-
- Electrospray ionization (ESI)
- Thermospray ionization (TSI)
- Atmospheric pressure chemical ionization (APCI)
- Atmospheric pressure photoionization (APPI)
- Particle beam ionization
Electron spray Ionization
In this technique, analyte ions are created in solution from the analyte before they are passed to the mass spectrometer.
The LC eluent is sprayed i.e nebulised in a chamber at atmospheric pressure in the presence of a strong electrostatic field and heated drying gas. The electrostatic field causes further dissociation of the analyte molecules. The heated drying gas causes the solvent in the droplets to evaporate. As the droplets shrink, the charge concentration in the droplets increases. Eventually, the repulsive force between ions with like charges exceeds the cohesive forces and ions are ejected(desorbed) into the gas phase. These ions are attracted to and passthrough a capillary sampling orifice into the mass analyzer.
Atmospheric pressure chemical ionization (APCI)
In APCI, the LC eluent is sprayed through a heated (typically 250 C –400°C) vaporizer at atmospheric pressure. The heat vaporizes the liquid. The resulting gas-phase solvent molecules are ionized by electrons discharged from a corona needle. The solvent ions then transfer their charge to the analyte molecules through various chemical reactions in the form of chemical ionization. The analyte ions pass through a capillary sampling orifice into the mass analyzer.
Atmospheric pressure photoionization(APPI).
Atmospheric pressure photoionization (APPI)for LC/MS is a relatively new technique. As in APCI, a vaporizer converts the LC eluent to the gas phase. A discharge lamp generates photons in a narrow range of ionization energies. The range of energies is carefully chosen to ionize as many analyte molecules as possible while minimizing the ionization of solvent molecules. The resulting ions passthrough a capillary sampling opening or orifice into the mass analyzer.
Particle Beam Ionization
A fast particle bean is directed onto the analyte and matrix as a sample. These are ionised into either analyte or matrix ions where they are carried on a secondary ion bean through an extraction grid and then to the mass spectrometer.
Mass Analyser
The mass of an ion is measured by deflecting ions down a curved tube in a magnetic field. The size is based on the level of deflection. This deflection is a function of their kinetic energy determined by the mass, charge and velocity.
The magnetic field is scanned to measure different ions.
Types of mass analyzer:- (1) Quadrapole mass filter. (2) time of flight (3) Ion trap (4) Fourier transform ion cyclotron resonance (FT-ICR or FT-MS).
Quadrapole Mass Filters
A quadrupole mass analyzer consists of four parallel rods arranged in a square. The analyte ions are directed down the center of the square.
Voltages applied to the rods generate electromagnetic fields. These fields determine which mass-to-charge ratio of ions can pass through the filter at a given time.
Quadrupoles tend to be the simplest and least expensive mass analyzers. Quadrupole mass analyzers can operate in two modes:
•Scanning (scan) mode
• Selected ion monitoring (SIM) mode.
In scan mode, the mass analyzer monitors a range of mass-to-charge ratios.
In SIM mode, the mass analyzer monitors only a few mass to-charge ratios. SIM mode is significantly more sensitive than scan mode but provides information about fewer ions. Scan mode is typically used for qualitative analyses or for quantitation when all analyte masses are not known in advance. SIM mode is used for quantitation and monitoring of target compounds.
Time Of Flight
In a time-of-flight (TOF) mass analyzer, a uniform electromagnetic force is applied to all ions at the same time, causing them to accelerate down a flight tube.
Lighter ions travel faster and arrive at the detector first, so the mass-to-charge ratios of the ions are determined by their arrival times. Time-of flight mass analyzers have a wide mass range and can be very accurate in their mass measurements.
Ion trap
An ion trap mass analyzer consists of a circular ring electrode plus two end caps that together form a chamber. Ions entering the chamber are “trapped” there by electromagnetic fields. Another field can be applied to selectively eject ions from the trap. Ion traps have the advantage of being able to perform multiple stages of mass spectrometry without additional mass analyzers.
Fourier transform ion cyclotron resonance (FT-ICR or FT-MS)
An FT-ICR mass analyzer (also called FT-MS) is another type of trapping analyzer. Ions entering a chamber are trapped in circular orbits by powerful electrical and magnetic fields. When excited by a radio-frequency (RF) electrical field, the ions generate a time dependent current. This current is converted by Fourier transform into orbital frequencies of the ions which correspond to their mass-to charge ratios.
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