LIQUID CHROMATOGRAPHY –MASS SPECTROSCOPY (LC-MS) Presented by Md Akbar Siddiq Khan M.Pharm Nizam College Of Pharmacy Hyderabad - A.P
What is LC-MS?• It is the combination of liquid chromatography and the mass spectrometry.• In LC-MS we are removing the detector from the column of LC and fitting the column to interface of MS.• In the most of the cases the interface used in LC- MS are ionization source.
PROBLEMS IN COMBINING HPLC AND MS HPLC MS• Liquid phase operation Vacuum operation• 25 - 50 deg. C 200 - 300 deg. C• No mass range Up to 4000 Da for quadrupole MS limitations Requires volatile buffers• Inorganic buffers Accepts 10 ml/min gas• 1 ml/min eluent flow is flow equivalent to 500 ml/min of gas
PARTS OF LC-MSTwo key components in this process are theion source, which generates the ions, and the mass analyzer, which sorts the ions. Several different types of ion sources are commonly used for LC/MS.
MOBILE PHASE:-The mobile phase is the solvent that moves the solute through out column. General requirements:- (1)low cost, uv transperancy,high purity. (2)low viscosity, low toxicity, non flammability. (3)non corrosive to LC system component. Solvent strength and selectivity:- it is the ability of solvent to elute solutes from a column.
COLUMN:- Column type:- Specialized mode:- The use of di-functional or tri-functional silanes to create bonded groups with two or three attachement points leading to phases with higher stability in low or higher pH and lower bleed for LCMS Most widely used columns for LCMS are:- (1) fast LC column. the use of short column. (15-50mm) (2) Micro LC column. the use of large column. ( 20-150mm)
Sample preparation:- Sample preparation generally consists of concentrating the analyte and removing compounds that can cause background ion or suppress ionization. Example of sample preparation include:- (1) on –column concentration to increase analyte concentration. (2) desalting to reduce the sodium and potassium adduct formation that commonly occurs in electro spray. (3) filtration to separate a low molecular-weight drug from proteins in plasma, milk, or tissue.
Ionization and Interface:-• It is difficult to interface a liquid chromatography to a mass-spectrometer cause of the necessity to remove the solvent.• The commnly used interface are:- (1) Electrospray ionization (ESI) (2) Thermospray ionization (TSI) (3) Atmospheric pressure chemical ionization (APCI) (4) Atmospheric pressure photoionization(APPI) (5) Partical beam ionization.
Electron spray ionization.Generate analyte ions in solution beforethe analyte reaches the massspectrometer.The LC eluent is sprayed (nebulized) intoa chamber at atmospheric pressure in thepresence of a strong electrostatic field andheated drying gas.The electrostatic field causes furtherdissociation of the analyte molecules.The heated drying gas causes the solventin the droplets to evaporate. As the dropletsshrink, the charge concentration in thedroplets increases.Eventually, the repulsive force betweenions with like charges exceeds thecohesive forces and ions are ejected(desorbed) into the gas phase.These ions are attracted to and passthrough a capillary sampling orifice into themass analyzer.
Atmospheric pressure chemical ionization (APCI)In APCI, the LC eluent is sprayedthrough a heated (typically 250 C –400 C) vaporizer at atmosphericpressure.The heat vaporizes the liquid. Theresulting gas-phase solventmolecules are ionized by electronsdischargedfrom a corona needle.The solvent ions then transfercharge to the analyte moleculesthrough chemical reactions(chemical ionization).The analyte ions pass through acapillary sampling orifice into themass analyzer.
Atmospheric pressure photoionization(APPI)Atmospheric pressurephotoionization (APPI)for LC/MS is arelatively new technique. As in APCI, a vaporizer convertsthe LC eluent to the gas phase. Adischarge lamp generates photons ina narrow range of ionization energies. The range of energies is carefullychosen to ionize as many analytemolecules as possible whileminimizing the ionization of solventmolecules.The resulting ions passthrough acapillary sampling orifice into themass analyzer.
Mass Analyser:-• They deflects ions down a curved tubes in a magnetic fields based on 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) modeIn scan mode, the mass analyzer monitors a range of mass-to-charge ratios.In SIM mode, the mass analyzer monitors only a few massto-charge ratios. SIMmode is significantly more sensitive than scan mode but provides information aboutfewer ions. Scan mode is typically used for qualitative analyses or for quantitationwhen all analyte masses are not known in advance.SIM mode is used for quantitationand monitoring of target compounds.
time of flightIn a time-of-flight (TOF) massanalyzer, a uniformelectromagnetic force is applied toall ions at the same time, causingthem to accelerate down a flighttube.Lighter ions travel faster andarrive at the detector first, so themass-to-charge ratios of the ionsare determined by their arrivaltimes.Time-offlight mass analyzershave a wide mass range and canbe very accurate in their massmeasurements.
Ion trapAn ion trap mass analyzer consists of a circular ring electrode plus twoend caps that together form a chamber. Ions entering the chamber are“trapped” there by electromagnetic fields. Another field can be applied toselectively eject ions from the trap.Ion traps have the advantage of being able to perform multiple stages ofmass 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 andmagnetic fields.When excited by a radio-frequency (RF) electrical field, the ions generate atimedependent current.This current is converted by Fourier transform into orbital frequencies of the ions whichcorrespond to their mass-tocharge ratios.
Molecular weight determination Determining the molecular weight of green fluorescent proteins Structural determination e.g. structural determination of ginsenoside. Pharmaceutical application e.g. identification of bile acids metabolites. Biochemical application e.g. rapid protein identification using capillary lc/ms/ms. Food application e.g. identification of aflatoxin in food determination of vitamin D3 in poultry feed supplement using MS3 Environmental application e.g. detection of phenyl urea herbicides, detection of low level of carbaryl in food.
Referance:-• Wikimedia Foundation. 2010.(1) W. Paul & H. Steinwedel; Zeitschrift für Naturforschung, 8A; 1953, p448.(2) W. Paul; Agewandte Chemie - International Edition, 29; 1990, p739.(3) G. C. Stafford et al.; International Journal of Mass Spectrometry and Ion Processes, 60; 1984, p85 and Analytical Chemistry, 59; 1987, p1677.