AN OVERVIEW OF LC-MS AND GC-MS ITS APPLICATION

SUBMITTED BY NIHAL. P FIRST YEAR M.PHARM NATIONAL COLLEGE OF PHARMACYUNDER GUIDENCE OF PROFF.KATHIRVEL

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* 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

LC-MS:

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PARTS OF LC-MS

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INTERFACEApart from being an inlet system for theMS, an LC–MS interface is also the couplingof a detector (MS) to a chromatograph.The choice of LC–MS interface stronglyinfluences the characteristics of the MS asa detector for LC. Therefore, we shouldkeep in mind what characteristics are idealfor an LC detector

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1. Direct liquid introduction (DLI):

- The DLI interface was developed in order to solve the problems with in-

capillary evaporation in the capillary inlet.

- In a DLI interface, the column effluent is nebulized by the disintegration

into small droplets of a liquid jet formed at a small diaphragm After

desolvation of the droplets in a desolvation chamber, the analytes can be

analysed using solvent-mediated CI with the LC solvents as the reagent

gas.

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Advantages:

• No heat is applied to the interface and it is therefore able to deal with thermally

labile materials better than the moving-belt interface.

• The interface contains no moving parts and is cheap and simple to construct

and operate and is inherently more reliable than the moving-belt interface.

• Both positive- and negative-ion CI spectra can be generated and the interface

provides molecular weight information, plus it can also be used for sensitive

quantitative and semi-quantitative procedures.

Disadvantages:

• Involatile compounds are not usually ionized with good efficiency.

• The pinhole is prone to blockage and therefore the system must be kept

completely free of solid materials.

• Only a small proportion of the flow from a conventional HPLC column is able to

enter the source of the mass spectrometer and sensitivity is consequently low.

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2. Moving belt/wire:

• In a moving-belt interface (MBI), the column effluent is deposited onto an

endless moving belt from which the solvent is evaporated by means of gentle

heating and efficiently exhausting the solvent vapours. After removal of the

solvents, the analyte molecules are (thermally) desorbed from the belt into the

ion source and mass analysed.

• The MBI for LC.MS was used in a wide variety of applications, including the

analysis of drugs and their metabolites, pesticides, steroids, alkaloids,

polycyclic, aromatic hydrocarbons and others.

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Advantages:

• The interface can be used with a wide range of HPLC conditions.

• The analyst does have some choice of the ionization method to be used; EI,

CI and FAB are available, subject to certain limitations, and thus both molecular

weight and structural information may be obtained from the analyte(s) under

investigation.

Disadvantages:

• An intense chemical background from the material from which the belt was

made is often observed in the mass spectra generated by this type of interface

unless adequate conditioning is carried out.

• The belt is prone to break during operation.

• Problems may be encountered in the analysis of thermally labile compounds,

as heat is required for mobile-phase removal and for the transfer of analyte

from the belt into the source of the mass spectrometer, and highly involatile

compounds which cannot be desorbed from the belt, unless FAB is used for

ionization.

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3. Thermospray (TSP):

They are of 2 type:

a) Real-TSP ionization

b) Discharge electrode for external ionization and repeller electrode

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• In a thermospray (TSP) interface, a jet of vapour and small droplets is formed

out of a heated vaporizer tube into a low-pressure region. Nebulization takes

place as a result of the disruption of the liquid by the expanding vapour that is

formed upon evaporation of part of the liquid in the tube.

• Before the onset of the partial inside-tube evaporation a considerable amount

of heat is transferred to the solvent, which assists in the desolvation of the

droplets in the low pressure region. By applying efficient pumping directly at the

ion source up to 2 ml/min of aqueous solvents can be introduced into the MS

vacuum system. The ionization of the analytes takes place by means of ion-

molecule reactions and ion evaporation processes.

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4. Partical beam (PB):

• During the desolvation of the droplets, the less volatile analyte molecules

coagulate into small particles, typically 50-300nm. The solvent vapour and

nebulization gas are separated from the particles by means of molecular-beam

technology.

• The mixture is expanded at a nozzle into a vacuum chamber. The low mass

solvent molecules show a greater tendency to diffuse away from the centre of

the expansion, while the heavier analyte particles are kept in the core of the

vapour jet. The core of the jet is then sampled by a skimmer.

• The region between the first set of orifices is maintained at between 2 and 10

torr, and that between the second set between 0.1 and 1 torr. In the momentum

separator, sufficient pressure reduction is achieved to generate EI and solvent-

independent CI mass spectra.

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5. Atmospheric pressure chemical ionization (APCI)

• Here, the HPLC effluent is passed through a pneumatic nebulizer where the

droplets are both generated and desolvated. The spray so formed then passes

through a heated region where the vapour is dried.

• The neutral species thus produced are then passed through a corona discharge –

the latter occurs when the field at the tip of the electrode is sufficiently high to ionize

the gas surrounding it but insufficiently high to cause spark – where ionization of the

analyte is effected by CI-type processes with the vaporized solvent acting as the

reagent gas.

• The technique is capable of dealing with flow rates between 0.5 - 2 ml/min, so

making it directly compatible with 4.6 mm columns, and is much more tolerant to a

range of buffers.

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6. Conti. Flow fast atom bombardment (cf-FAB)

• In a continuous-flow fast-atom bombardment (Cf-FAB) interface, typically a

5-15 µl/min liquid stream, mixed with 5% glycerol (or) thioglycerol (or)

nitrobenzyl alcohol as FAB matrix, flows through a narrow-bore fused-silica

capillary towards either a stainless-steel frit or a (goldplated) FAB target. At the

target or frit, a uniform liquid film is formed due to a subtle balance between

solvent evaporation and solvent delivery. Ions are generated by bombardment

of the liquid film by fast atoms or ions, common to FAB.

• The fast atom like Ar or Xe is used for bombarding the sample and ions are

sputtered out of the solution and enters into MS.

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APPPLICATION OF LC-MS

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.

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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.

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The sample solution is injected into the GC inlet where it is vaporized and swept onto a chromatographic column by the carrier gas (usually helium).

The sample flows through the column and the compounds comprising the mixture of interest are separated by interaction with the coating of the column (stationary phase) and the carrier gas (mobile phase).

The latter part of the column passes through a heated transfer line and ends at the entrance to ion source where compounds eluting from the column are converted to ions.

PRINCIPLE OF GC-MS

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COMPONENTS

The GC-MS is composed of two major building blocks: the gas chromatograph and the mass spectrometer.

Carrier Gas, N2 or He, 1-2 mL/min

Injector Oven Column Detector

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JET SEPARATOR

Two capillary tubes aligned with a small space between them. (1 mm)

A vacuum is created between the two tubes using a rotary pump.

The GC effluent enters the vacuum region, those molecules which continue in the same direction enter the second capillary tube and continue to the ion source.

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The carrier gas molecules are more easily diverted from the linear path by collisions.

The analyte molecules are much larger and carry more momentum.

The surface of the separator must be inactive and a reasonably even temperature.

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Jet Separator

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APPLICATIONS

Environmental Monitoring and Cleanup

GC-MS is becoming the tool of choice for tracking organic pollutants in the environment.

GC-MS can analyze the particles from a human body in order to help link a criminal to a crime. The analysis of fire debris using GC-MS is well established, and there is even an established American Society for Testing Materials (ASTM) standard for fire debris analysis.

Criminal forensic

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REFERENCES

1) PHARMACEUTICAL CHEMISTRY 4TH EDITION ,EDITED BY AH BECKETT AND JB STENLAKE.

2) GAS CHROMATOGRAPHY-MASS SPECTROSCOPY FROM WIKIPEDIA ,THE FREE ENCYCLOPEDIA.

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