1. PRINCIPLE, APPLICATIONS & ADVANCES IN
MASS SPECTROMETRY.

2. Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles.
It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and other chemical compounds .

3. Principle
A mass spectrum is obtain by converting component of sample into rapidly moving gaseous ions and resolving them on the basis of their mass to charge ratio.
Each kind of ion has a particular ratio of mass to charge ,i.e. m/e ratio.
For most ions , the charge is one and thus m/e ratio is simply the molecular mass of the ion.

4. The molecular ion is called parent ion & is designated as M+
The molecular ion is called parent ion & is designated as M+. it is positively charged molecule with an unpaired electron.
The set of ions are analyzed in such a way that a signal is obtained for each value of m/e that is represented. The intensity of each signal represent the relative abundance of the ion producing the signal.
The largest peak in the structure is called the base peak and its intensity is taken as 100%.

5. Involved following steps
1. A sample is loaded onto the MS instrument, and undergoes vaporization.
2. The components of the sample are ionized by one of a variety of methods which results in the formation of charged particles (ions).
3. The ions are separated according to their mass-to- charge ratio in an analyzer by electromagnetic fields.

6. 4. The ions are detected, usually by a quantitative method
5. The ion signal is processed into mass spectra .

7. Typical Mass Spectrum Characterized by sharp, narrow peaks.
X-axis position indicates the m/e ratio of a given ion (for singly charged ions this corresponds to the mass of the ion).
Y axis indicates the relative abundance of ions.

8. Mass spectra

9. Resolving Power
Width of peak indicates the resolution of the MS instrument.
The better the resolution or resolving power, the better the instrument and the better the mass accuracy.
Resolving power is defined as: M / (DM)
M is mass of the particle & (DM) is the difference in mass of 2 neighboring peak of equal intensity.

10. Types of ions produce in mass spectrometry -
Molecular ion ( parent ion )-
Mass spectra first of all display the molecular ion (or parent ion) peak which is a radical cation M+. as a result of removing one electron from the molecule .
eg- toluene the molecular ion peak is located at 92 m/e corresponding to its molecular mass.

11. 2. Fragment ion- E.g.- alcohol-
The excited molecular ions undergo decompositions to give rise to variety of fragments ion .
E.g.- alcohol-

12. 3. Rearrangement ion-
Fragment ion formed by the intramolecular rearrangement involving migration of hydrogen atoms from one part of the ion to Another are called rearrangement ions.
MacLafferty rearrangement-
It involves the migration of gamma hydrogen followed by cleavage of beta bond and leads the elimination of neutral molecule.

13. 4. Metastable ions-
The ions resulting from the decomposition between the source region and the magnetic analyzer are called metastable ions.
Which appear in the spectrum as broad peak.
5.multiple charged ion.
6. negative ion.

14. Advances in mass spectrometry
Although ordinary mass spectrometry is a powerful tool for identification & analysis of pure compound, & simple mixture.
But for complex mixture & interpretation of complex spectrum advances in mass spectrometry has been done.
For increasing accuracy ,selectivity.

15. Various advances in mass spectrometry
1. chromatography – mass spectrometry
a. gas chromatography – mass spectrometry (GC/MS)
b. liquid chromatography – mass spectrometry (LC/MS)
2. tandem mass spectrometry (ms-ms )
3.capillary electrophoresis - mass spectrometry

16. a. gas chromatography / mass spectrometry-
Mass spectrometer is coupled with one of chromatographic method, called as hyphenated technique.
a. gas chromatography / mass spectrometry-
The GC-MS is composed of two major building blocks the gas chromatograph and mass spectrometer

17. Instrumentation of GC/MS-

18. Allow a much finer degree of substance identification than either unit used separately.
Sample source for mass spectrometry is eluant from gas chromatography & detector for gas chromatography is mass spectrometry.

19. General use-
1. Identification & quantitation of volatile & semi volatile organic compounds in complex mixture.
2. Determination of mol wt & elemental composition of unknown organic compound .
3. Structural determination of unknown organic compound.

20. Common application-
Quntitation of pollutant in drinking water & water waste.
Quantitation of drug & their metabolite in blood & urine for both pharmacological & forensic application.
Identification of unknown org compound in hazardous waste dump.
Identification of reaction product .
Identification of industrial product for quality control.

21. Limitation
1. only compound with vapour pressure exceeding abt torr can be analyzed by GC/MS.
2. Determination of positional substitution on aromatic ring is often difficult.
3. thermal degradation of component.

22. B. Liquid chromatography – mass spectrometry =( LC/MS ) OR ( HPLC / MS )
(LC/MS) separates compounds chromatographically before they are introduced to the ion source and mass spectrometer.
It differs from GC/MS in that the mobile phase is liquid, usually a mixture of water and organic solvents, instead of gas.
commonly, an electrospray ionization source is used in LC/MS. There are also some newly developed ionization techniques like laser spray.

24. very high sensitivity and selectivity.
Generally its application is oriented towards the general detection and potential identification of chemicals in the presence of other chemicals (in a complex mixture).
Applications-
1. pharmacokinetics-
E.g. how quickly drug cleared from hepatic blood flow ,& organ of body.
2.proteiomics-
3. drug development- in different stages like peptide mapping , natural product dereplication.

25. 2. Tandem mass spectrometry
Limitation
In urine analysis often fail to distinguish between specific metabolites.
2. Tandem mass spectrometry
Also known as MS/MS or MS2, involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.
Multiple round of mass spectrometry.
Eg .one mass analyzer can isolate one peptide from many entering a mass spectrometer.

26. A second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID).
A third mass analyzer then sorts the fragments produced from the peptides.

28. Application- 1. sequencing of peptide .
2. sequencing of oligosaccharide.

29. 3.capillary electrophoresis - mass spectrometry
it is a combination of liquid separation technique of capillary electrophoresis & detection technique of mass spectrometry.
Higher sensitivity and better compound identification with accurate mass measurements.
It has applications in basic research in proteomics and quantitative analysis of biomolecules as well 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.

30. References –
Instrumental methods of chemical analysis by Gurdeep R. Chatwal & Sham K. Anand ,Himalaya Publishing House ,page no
Organic spectroscopy by William Kemp ,third edition,
Instrumental analysis by Skoog, Holler, Crouch ,India edition ,page no
Elementary organic spectroscopy ,principle & applications by, Y.R. Sharma. page no
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