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PRINCIPLE, APPLICATIONS & ADVANCES IN PRINCIPLE, APPLICATIONS & ADVANCES IN

MASS SPECTROMETRY.MASS SPECTROMETRY.

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*Mass spectrometryMass spectrometry (MS) is an analytical (MS) is an analytical technique that measures the mass-to-technique that measures the mass-to-charge ratio of charged particles. charge ratio of charged particles.

*It is used for determining masses of It is used for determining masses of particles, for determining the elemental particles, for determining the elemental composition of a sample or molecule, and composition of a sample or molecule, and for elucidating the chemical structures of for elucidating the chemical structures of molecules, such as peptides and other molecules, such as peptides and other chemical compounds .chemical compounds .

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Principle Principle *A mass spectrum is obtain by converting A mass spectrum is obtain by converting component of sample into rapidly moving component of sample into rapidly moving gaseous ions and resolving them on the basis of gaseous ions and resolving them on the basis of their mass to charge ratio.their mass to charge ratio.

*Each kind of ion has a particular ratio of mass Each kind of ion has a particular ratio of mass to charge ,i.e. m/e ratio.to charge ,i.e. m/e ratio.

*For most ions , the charge is one and thus m/e For most ions , the charge is one and thus m/e ratio is simply the molecular mass of the ion.ratio is simply the molecular mass of the ion.

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1.1. The molecular ion is called parent ion & is The molecular ion is called parent ion & is

designated as M+. it is positively charged designated as M+. it is positively charged molecule with an unpaired electron.molecule with an unpaired electron.

2.2. The set of ions are analyzed in such a way The set of ions are analyzed in such a way that a signal is obtained for each value of m/e that a signal is obtained for each value of m/e that is represented. The intensity of each that is represented. The intensity of each signal represent the relative abundance of signal represent the relative abundance of the ion producing the signal.the ion producing the signal.

3.3. The largest peak in the structure is called the The largest peak in the structure is called the base peak and its intensity is taken as 100%.base peak and its intensity is taken as 100%.

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Involved following steps Involved following steps

1. 1. A sample is loaded onto the MS instrument, and undergoes vaporization.

2. The components of the sample are ionized by one 2. The components of the sample are ionized by one of a variety of methods which results in the of a variety of methods which results in the formation of charged particles (ions). formation of charged particles (ions).

3. The ions are separated according to their mass-3. The ions are separated according to their mass-to-charge ratio in an analyzer by to-charge ratio in an analyzer by electromagnetic fields. electromagnetic fields.

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4. The ions are detected, usually by a quantitative 4. The ions are detected, usually by a quantitative method method

5. The ion signal is processed into mass spectra5. The ion signal is processed into mass spectra . .

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Typical Mass SpectrumTypical Mass Spectrum

*Characterized by sharp, narrow peaks.Characterized by sharp, narrow peaks.

*X-axis position indicates the m/e ratio X-axis position indicates the m/e ratio of a given ion (for singly charged ions of a given ion (for singly charged ions this corresponds to the mass of the this corresponds to the mass of the ion).ion).

*Y axis indicates the relative Y axis indicates the relative abundance of ions.abundance of ions.

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* Resolving PowerResolving Power*Width of peak indicates the resolution of the Width of peak indicates the resolution of the MS instrument.MS instrument.

*The better the resolution or resolving power, The better the resolution or resolving power, the better the instrument and the better the the better the instrument and the better the mass accuracy.mass accuracy.

*Resolving power is defined as: M / (Resolving power is defined as: M / (M)M)

M is mass of the particle & (M is mass of the particle & (M) is the M) is the difference in mass of 2 neighboring peak of difference in mass of 2 neighboring peak of equal intensity.equal intensity.

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*Types of ions produce in Types of ions produce in mass spectrometry -mass spectrometry -

1.1. Molecular ion ( parent ion )-Molecular ion ( parent ion )-

Mass spectra first of all display the Mass spectra first of all display the molecular ionmolecular ion (or (or parent ionparent ion) peak ) peak which is a radical cation M+. as a result of which is a radical cation M+. as a result of removing one electron from the molecule .removing one electron from the molecule .

eg- toluene the molecular ion peak is eg- toluene the molecular ion peak is located at 92 m/e corresponding to its located at 92 m/e corresponding to its molecular mass. molecular mass.

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2. Fragment ion-2. Fragment ion-

TheThe excited molecular ions undergo excited molecular ions undergo decompositions to give rise to variety of fragments decompositions to give rise to variety of fragments ion .ion .

E.g.- alcohol-E.g.- alcohol-

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3. Rearrangement ion- 3. Rearrangement ion-

Fragment ion formed by the intramolecular Fragment ion formed by the intramolecular rearrangement involving migration of hydrogen rearrangement involving migration of hydrogen atoms from one part of the ion to Another are atoms from one part of the ion to Another are called rearrangement ions.called rearrangement ions.

MacLafferty rearrangement-MacLafferty rearrangement-

*It involves the migration of gamma hydrogen It involves the migration of gamma hydrogen followed by cleavage of beta bond and leads the followed by cleavage of beta bond and leads the elimination of neutral molecule.elimination of neutral molecule.

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4. Metastable ions- 4. Metastable ions-

The ions resulting from the decomposition The ions resulting from the decomposition between the source region and the magnetic between the source region and the magnetic analyzer are called metastable ions.analyzer are called metastable ions.

Which appear in the spectrum as broad peak.Which appear in the spectrum as broad peak.

5.multiple charged ion.5.multiple charged ion.

6. negative ion.6. negative ion.

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*Advances in mass Advances in mass spectrometryspectrometry

*Although ordinary mass spectrometry is a Although ordinary mass spectrometry is a powerful tool for identification & analysis of pure powerful tool for identification & analysis of pure compound, & simple mixture. compound, & simple mixture.

* But for complex mixture & interpretation of But for complex mixture & interpretation of complex spectrum advances in mass spectrometry complex spectrum advances in mass spectrometry has been done.has been done.

*For increasing accuracy ,selectivity.For increasing accuracy ,selectivity.

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*Various advances in mass Various advances in mass spectrometryspectrometry

1. chromatography – mass spectrometry1. chromatography – mass spectrometry a. gas chromatography – mass a. gas chromatography – mass spectrometry (GC/MS)spectrometry (GC/MS)

b. liquid chromatography – mass b. liquid chromatography – mass spectrometry (LC/MS)spectrometry (LC/MS)

2. tandem mass spectrometry (ms-ms )2. tandem mass spectrometry (ms-ms )

3.capillary electrophoresis - mass 3.capillary electrophoresis - mass spectrometryspectrometry

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*1.Chromatography – mass spectrometry1.Chromatography – mass spectrometry

*Mass spectrometer is coupled with one of Mass spectrometer is coupled with one of chromatographic method, called as hyphenated chromatographic method, called as hyphenated technique.technique.

*a. gas chromatography / mass a. gas chromatography / mass spectrometry-spectrometry-

*The GC-MS is composed of two major building The GC-MS is composed of two major building blocks the gas chromatograph and mass blocks the gas chromatograph and mass spectrometerspectrometer

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* Allow a much finer degree of substance Allow a much finer degree of substance identification than either unit used identification than either unit used separately.separately.

* Sample source for mass spectrometry is Sample source for mass spectrometry is eluant from gas chromatography & eluant from gas chromatography & detector for gas chromatography is mass detector for gas chromatography is mass spectrometry.spectrometry.

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*General use-General use-11. Identification & quantitation of volatile & semi . Identification & quantitation of volatile & semi volatile organic compounds in complex mixture.volatile organic compounds in complex mixture.

2. Determination of mol wt & elemental 2. Determination of mol wt & elemental composition of unknown organic compound .composition of unknown organic compound .

3. Structural determination of unknown organic 3. Structural determination of unknown organic compound.compound.

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* Common application-Common application-1.1. Quntitation of pollutant in drinking water & water Quntitation of pollutant in drinking water & water

waste.waste.

2.2. Quantitation of drug & their metabolite in blood & Quantitation of drug & their metabolite in blood & urine for both pharmacological & forensic urine for both pharmacological & forensic application.application.

3.3. Identification of unknown org compound in Identification of unknown org compound in hazardous waste dump.hazardous waste dump.

4.4. Identification of reaction product .Identification of reaction product .

5.5. Identification of industrial product for quality Identification of industrial product for quality control.control.

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*LimitationLimitation

*1. only compound with vapour pressure exceeding 1. only compound with vapour pressure exceeding abt abt 1010-10 torr can be analyzed by GC/MS.-10 torr can be analyzed by GC/MS.

*2. Determination of positional substitution on 2. Determination of positional substitution on aromatic ring is often difficult.aromatic ring is often difficult.

*3. thermal degradation of component.3. thermal degradation of component.

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*B. Liquid chromatography – mass spectrometry B. Liquid chromatography – mass spectrometry =( LC/MS ) OR ( HPLC / MS )=( LC/MS ) OR ( HPLC / MS )

* (LC/MS) separates compounds chromatographically (LC/MS) separates compounds chromatographically before they are introduced to the ion source and before they are introduced to the ion source and mass spectrometer. mass spectrometer.

*It differs from GC/MS in that the mobile phase is It differs from GC/MS in that the mobile phase is liquid, usually a mixture of water and organic liquid, usually a mixture of water and organic solvents, instead of gas. solvents, instead of gas.

* commonly, an electrospray ionization source is used commonly, an electrospray ionization source is used in LC/MS. There are also some newly developed in LC/MS. There are also some newly developed ionization techniques like laser spray.ionization techniques like laser spray.

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*very high sensitivity and selectivity. very high sensitivity and selectivity.

* Generally its application is oriented towards the Generally its application is oriented towards the general detection and potential identification of general detection and potential identification of chemicals in the presence of other chemicals (in a chemicals in the presence of other chemicals (in a complex mixture). complex mixture).

*Applications-Applications-

1. pharmacokinetics-1. pharmacokinetics- E.g. how quickly drug cleared from hepatic blood E.g. how quickly drug cleared from hepatic blood flow ,& organ of body.flow ,& organ of body.

2.proteiomics-2.proteiomics- 3. drug development- in different stages like 3. drug development- in different stages like peptide mapping , natural product dereplication.peptide mapping , natural product dereplication.

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*LimitationLimitation*In urine analysis often fail to distinguish between In urine analysis often fail to distinguish between

specific metabolites.specific metabolites.

*2. Tandem mass spectrometry 2. Tandem mass spectrometry * AAlso known as lso known as MS/MSMS/MS or or MS2MS2, involves multiple , involves multiple

steps of mass spectrometry selection, with some form steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.of fragmentation occurring in between the stages.

*Multiple round of mass spectrometry.Multiple round of mass spectrometry.

*Eg .one mass analyzer can isolate one peptide from Eg .one mass analyzer can isolate one peptide from many entering a mass spectrometer. many entering a mass spectrometer.

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*A second mass analyzer then stabilizes the A second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation them to fragment by collision-induced dissociation (CID). (CID).

*A third mass analyzer then sorts the fragments A third mass analyzer then sorts the fragments produced from the peptides. produced from the peptides.

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*Application-Application-*11. sequencing of peptide .. sequencing of peptide .

*2. sequencing of oligosaccharide.2. sequencing of oligosaccharide.

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3.capillary electrophoresis - 3.capillary electrophoresis - mass spectrometrymass spectrometry *it is a combination of liquid separation technique it is a combination of liquid separation technique

of capillary electrophoresis & detection technique of capillary electrophoresis & detection technique of mass spectrometry.of mass spectrometry.

*Higher sensitivity and better compound identification with accurate mass measurements.

*It has applications in basic research in proteomics It has applications in basic research in proteomics and quantitative analysis of biomolecules as well and quantitative analysis of biomolecules as well as in clinical medicine.as in clinical medicine.

*The applicability of CE-MS is demonstrated on a separation of glycopeptides from monoclonal antibodies (mAb) and to identify the glycan modifications.

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* References –References –

1.1. Instrumental methods of chemical analysis by Instrumental methods of chemical analysis by Gurdeep R. Chatwal & Sham K. Gurdeep R. Chatwal & Sham K. Anand ,Himalaya Publishing House ,page no. Anand ,Himalaya Publishing House ,page no. 2.272-2.302.2.272-2.302.

2.2. Organic spectroscopy by William Kemp ,third Organic spectroscopy by William Kemp ,third edition, 285-306.edition, 285-306.

3.3. Instrumental analysis by Skoog, Holler, Instrumental analysis by Skoog, Holler, Crouch ,India edition ,page no. 498- 533. Crouch ,India edition ,page no. 498- 533.

4.4. Elementary organic spectroscopy ,principle & Elementary organic spectroscopy ,principle & applications by, Y.R. Sharma. page no. 280-291.applications by, Y.R. Sharma. page no. 280-291.

5.5. www. google .com.www. google .com.

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