GCMS

GAS CHROMATOGRAPHY-GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MASS SPECTROMETRY (GC-

MSMS))Presented byAPARNA.T

UNDER THE GUIDANCE OF

Mr. Ch. DEVADASU M.Pharm.,

Assistant professor

Department of PA & QA

VIGNAN PHARMACY COLLEGE (Approved by AICTE, PCI &

Affiliated to JNTU-K)VADLAMUDI, 522213.

13/9/2014VIGNAN PHARMACY COLLEGE

IUPAC: chromatography is a physical method of separation in

which the components to be separated are distributed between

two phases. One of which is stationary (stationary phase) while

the other (the mobile phase) moves in a definite direction.

Elution chromatography is a procedure in which the mobile

phase is continuously passed through or along the

chromatographic bed and the sample is fed into the system as a

finite slug. EX: Gas Liquid Chromatography(GC)

Gas chromatography is a separation method in which the

components of a sample partition between two phases one of

these phases is a stationary bed with a large surface area, and

the other is a gas which percolates through the stationary bed.13/9/2014 2VIGNAN PHARMACY COLLEGE

The father of modern gas chromatography is Nobel Prize

winner John Porter Martin, who also developed the first

liquid-gas chromatograph. (1950)

13/9/2014 3VIGNAN PHARMACY COLLEGE



In GC the main principle of separation is partition.

1. A gaseous mobile phase flows continuously

through the column which is coated with a liquid

stationary phase.

2. The sample is introduced into the heated injection

port where it is vaporized and carried in to the

column.

3. The sample partition between the mobile phase

and stationary phase, and is separated in to

individual components based on relative solubility in

liquid stationary phase at the given temperature.

4. The components of the sample separate from one

another based on their relative vapor pressures and

affinities for the stationary bed.

5. .


THE CHROMATOGRAPHIC PROCESS - PARTITIONINGTHE CHROMATOGRAPHIC PROCESS - PARTITIONING

(gas or liquid)

MOBILE PHASEMOBILE PHASE

STATIONARY PHASESTATIONARY PHASE

Sampleout

Samplein

(solid or heavy liquid coated onto a solid or support system)


In the animation below the red molecules are more soluble in the liquid (or less volatile) than are the green molecules.


Definition:

Different affinity of any 2 components to stationary phase causes the separation.

Concentration of component A in stationary phase

Concentration of component A in mobile phase

Distribution Coefficient



G. C is a separation technique.

Small amounts of sample for example 1 ml of air,1

micro lit. of the solution either liquids and solids in

solution are injected in to an Instrument .

The machine is called a Gas chromatograph

this machine by using injection port, column and

detector generates a written record of analysis .. a

series of peaks . Series of peaks are called a

chromatogram.

Chromatogram is simply a written record of the

analysis performed by the gas chromatograph.



The concept of mass spectrometry was first put forth by Sir J.J Thomson, English Physicist Who discovered the

electron in 1887.He got 1906 Nobel Laureate in Physics.

DEMPSTER

Sir J.J Thomson


What does a mass spectrometer do?Mass –spec or simply MS is a super important technique

Mass spec is easy technique to give you Molecular weight

(from molecular ion (M+)

You can get Molecular formula (Elements present).

Nearly ALL ELEMENTS in the periodic table can be determined by

mass spectrometry.

MS is incredibly valuable in getting structure (from fragments) of Bio

molecules such as peptides and proteins and also

natural products and also organic structures.

It can give information about chemical structures.




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The mass spectrometer is an instrument which help in separating the individual

atoms or molecules because of difference in their masses.

Consider a molecule M, Which is bombarded with a beam of electrons

M + e- M+. +2e-

where, M+. is molecular ion or radical ion

2e- is electron

Now voltage “v” is applied in an electric field then ions are accelerated. In this

condition the energy given to each particle is zV and this is equal to kinetic

energy which is equal to 1/2mv2 .

THEORY


i.e. potential energy=kinetic energy

zV = 1/2mv2

2zV = mv2

2zV/m= v2

= v

Where V = Velocity of particle

m = mass

z = charge of an electron

V = Acceleration voltage

All the particles posses some energy zV with some kinetic energy

1/2mv2, but m value changes from molecule to molecule with respect velocity

‘v’ also changes. i.e. ½ mv=zV1813/9/2014VIGNAN PHARMACY COLLEGE

When all charged particles have been accelerated by an applied voltage, they

enter into a magnetic field “H”. Then attractive force is HzV. And balancing

force of particle is mv2 /r.

Centripetal = Centrifugal

HzV=mv2/r

Hz=mv/r

From the above equation v=

Hz=m / r

By squaring on both sides

H2 z2 = m2 (2zV/m) / r2

H2 z = 2v m/ r2

m/z = H2 r2 / 2v 1913/9/2014VIGNAN PHARMACY COLLEGE

MASS SPECTRUM

The mass spectrum is the plot of mass to charge ratio of positively charged

ions against their relative abundance. The m/z ratio are taken along the

abscissa, while relative abundance is taken on ordinate.

BASE PEAK:

The most intense peak in the mass spectrum is called the base peak. Base

peak is the highest peak it is assigned a relative intensity of 100%.


MOLECULAR ION PEAK:

The ion formed from a molecule by removal of one electron of lowest

ionization potential is known as molecular ion.

The molecular ion is detected as mass to charge ratio that corresponds to

molecular weight of molecule. The molecular ion peak gives the molecular

weight of compound . The molecular ion peak is highest mass number except

isotope peak. Molecular ion peakBase peak

Fragment ions


FRAGMENT IONS:

The ions produced from the molecular ion by cleavage of bonds are called

fragment ions

They have lower masses and used as building blocks to reconstruct the

molecular structure. Fragmentation of molecular ion cleavage bond occurs in

heterolytic and homolytic cleavage.

METASTABLE IONS:

Mass spectrum of molecule shows sharp peaks at m/z integrals. But some show

diffuse, broad low intensity peaks at non integral m/z values these are called

metastable ions

m1+ m2

++ neutral fragment


If in the reaction m1+--------->m2

++ takes place in source then the

daughter ion may be m2+. But m1

+----->m2++ if occurs after the source and

before arrival at collector at lower mass than m2+ and is said to be

metastable ion m*.The peak (m*) due to such fragmentation therefore

occurs at lower mass than m2+ and generally broad. The relation between

the m* with that of m1+ & m2+ can be written as

m*=(m2)2/m1


Rela

tive a

bu

nd

an

ce

m/z values

110108

8179

29 –C2 H5

M+2

M+

CH3CH2Br


Gas chromatography–mass spectrometry (GC-MS, or

alternatively HPLC-MS) is an ADVANCED ANALYTICAL

INSTRUMNTAL technique that combines the physical

separation capabilities of GAS CHROMATOGRAPHYGAS CHROMATOGRAPHY with

the mass analysis capabilities of MASS SPECTROMETER

13/9/2014VIGNAN PHARMACY COLLEGE 26

27

Gas

chromatography

Mass spectrometry

GC-MS

Separates mixture of

components into

individual

Identifies molecules

based on their mass

A chemical analysis

technique combining two instruments to

provide for powerful

separation & identification.


Coupling of GC to MS:GC

Atmospheric density heated

(200-300 ∘C)

Interfaces

MSHigh

vacuum (10-

6 torr)heated

The interface b/w the GC&MS is an

important role to play in the overall efficiency

of the instrument.

Both system are heated (200 -300 ∘C) both

deal with compounds in the vapor state.Only one problem is that the atmospheric

pressure output of the GC must be reduced to

vacuum of

10-5 – 10-6 torr for the MS inlet

Types of interfaces:


Capillary direct interface:

Today most GC-MS systems use capillary columns &

fused silica tubing permits an inert,high efficiency,direct

transfer between the 2 systems.

Flow rates is 5ml/min.

Capillary direct interface:

Today most GC-MS systems use capillary columns &

fused silica tubing permits an inert,high efficiency,direct

transfer between the 2 systems.

Flow rates is 5ml/min.


Jet separator (packed column):

•The separator consist of two glass tubes aligned with a

Small distance between them.

•Carrier gas entering from the GC column is pumped away

by a separate vacuumed system.

•The larger sample molecules maintain their momentum

&pass preferentially in to the second capillary.

•Sample enrichment occurs & the initial atmospheric

pressure is reduced.


Bothe T & P surfaces activity of the glass jet separator must be controlled.


Watson & Biermann effusion separator:

• It consists of a sintered glass tube .

• The carrier usually Helium, passes preferentially

through the sintered glass tube & the effluent in

concentrated by a factor of up to 100.

• The gas flow rates in the order of 20-60ml/min.


It converts the components of a sample into ions

by bombardment with electrons, ions, molecules.

IONIZER;

CH3OH + 1e CH3OH+ + 2e

molecular ion or radical ion

The gas molecules exiting the GC are bombarded

by a high energy electron beam.


An electron which sticks a molecule may impart

enough energy to remove another electron from

that molecule.

The charged molecule is known as molecular ion.

The molecular ion can causes that ion to break

into smaller pieces.

CH3OH+ CH2OH+ + H-



The most common form of ionization is EI.

Electrons are produced by tungsten filament.

These electrons accelerated towards the ion source

chamber.

The electrons require an energy equal to the voltage

B/W the filament & ion source chamber. 70 ev is commonly used.

A proportion of electron beam will strike the electron

trap producing trap current.



A permanent magnet is positioned across the ion

chamber to produce a magnetic flux in parallel to the

electron beam.

A (+)ve ion repelle voltage & (-)ve ion excitation

voltage works to gather to produce an electric field in

the source chamber.

Such that ions leaves through ion exit slit.

The ions are directed through the various focusing &

centering lenses are focused on to the source exit slit.


In CI a reagent gas methane or ammonia or

isobutene are introduced into the mass

spectrometer.

The reagent gas will interact with the electron to

produce radical electrons.

EG;

R + e R+ + 2e



It is a good for organic compounds.

Usually produces (M + H)+ (M+ CH3)+ adducts.

Adducts are not always abundant.

ISOBUTANE

Usually produces (M + H)+ ,(M+C4H9)+ adducts &

some fragmentation.

Adducts are more abundant than for methane CI .


Fragmentation is absent.

Polar compounds produces ( M+NH4)+ adducts.

Basic compounds produces (M+H)+ adducts.

Non polar, non basic compounds are not ionized.


44

Ionization method

electron impact

Chemical ionization

Typical analyses Relatively small volatile

Relatively small volatile

Sample introduction

GC (or) liquid/solid probe

GC/Liquid /solid probe

Mass range 1-1000 Dolton's 1-1000 Daltons

Method highlights

Hard method versatile provides structure

information

Soft method molecular ion peak (M+H)+


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Negative ion chemical ionization(NICI):

In NICI a reagent gas is used & the electrons collide with

it so that their energies are reduced to 10Ev.

Molecules with a high affinity for electrons are able to

capture these low energy thermal electrons.

This is known as NICI but it does not involved in the

formation of a chemical adduct.


46

AB+ e

AB-

Resonance electron capture

A- +BDissociative

electron capture.


47

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.


48

In a quadrupole mass analyser a set of four rods are arranged parallel to

the direction. Here a DC current and radio frequency RF is applied to

generate oscillating electrostatic field in between the rods. Based on this

only m/z is been determined and stable oscillation takes place. And ion

travels in quadrupole axis with cork screw type of trajectory.


49

TOF mass analyser is based on simple idea that the velocities of

two ions are created by uniform electromagnetic force applied to

all the ions at same time, causing them to accelerate down a flight

tube.

Lighter ions travels faster and strike the detector first so that the

m/z ratio of ions is detected.



The ion trap mass analyser operates by similar principles where it

consists of circular ring electrode

Plus two end caps that form a chamber. Here AC or DC power

along RF potential is applied between the cups and the ring

electrode.

There the ions entering into the chamber are trapped by

electromagnetic fields and they oscillates in concentric

trajectories. This process is called resonant ejection.



DATA HANDLING

All the mass spectrometers now employ computer control of same functions

and also use a computerised display and output.

The amount of data generated even by a fairly modest mass spectrometer is

very large indeed, a single run may store data for upto 100 fragments from

each type of molecule and if, GCMS analyses is being performed, a complete

mass spectrum is generated and stored every sec for upto 90 min


54

LIMITATIONS


•Only Compounds with Vapour Pressure exceeding about 1010 torr

can be analyzed b as chromatography –mass spectrometry.

•Certain isomeric compounds cannot be distinguished by mass

spectrometry (EG : naphthalene vs. azulene).


Elucidation of the structure of organic & biological molecules.

Impurity profiling of pharmaceuticals.

Identification of components in thin layer & paper chromatograms.

Identification of drugs of abuse & metabolites of drugs of abuse in blood,

urine & saliva.

Testing for the presence of the drugs in blood in race horses & in Olympic

athletic (in forensic GC-MS).

Analyzer of aerosol particles.

Determination of pesticide residues in food.

Polymer characterization (pyrolysis methods combined GCMS).

Drug monitoring & toxicology studies.

Determination of amino acid sequence in sample of polypeptides &

proteins.





The TIC GC chromatogram for Psoralen and the EI spectrum obtained from the chromatographic peak


EI CHLOROQUIN

NICI OF CHLOROQUIN


Impurities can arise from

•Manufacturing process

•Degradation of drug substanceIf impurities are greater than 0.1% concentration in drug substancethe name and structure of impurity is to be submitted to regulatory agencies.USFDA CONSIDERATIONS:IMPURITIES SHOULD BE LESS THAN 1.0%Presence of impurities must be illustrated with GC-MS/LC-MS chromatograms



1-naphthol





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.

GC-MS especially useful here as samples often contain very

complex matrices &results used in court.


GC-MS used for detection of illegal narcotics & may

eventually supplant drug-sniffing dogs.

It’s also commonly used in forensic toxicology to find drugs

&poisons in biological specimens of victims .


GC-Ms is main tool used in sports anti doping

laboratories to test athletes urine samples for

prohibited performance enhancing drugs.

EG : anabolic steroids.


Food & beverage contain numerous aromatic compounds ,

some naturally present in the raw materials &some forming

during process.

GC-MS is extensively used for the analysis of these

compounds which include ester, fatty acids , alcohols,

aldehydes, terpenes etc……


GC-MS 2 were brought to mars by the Viking program.

Venera11&12 pioneer Venus analyzed the atmosphere of

Venus with GC-MS.

The material in the comet 67p will be analyzed by the rosetla

mission with a chiral GC-MS in 2014.


In born errors of metabolism are now detectable by new born

screening tests, especially the testing using GC-MS .

It can determine compounds in urine even in minor

concentration.

The measurement of c13-c12 ratio with an isotope ratio mass

spectrometer.


GC-MS is one of the best analytical tool in GC-MS is one of the best analytical tool in

analysing the most of the compounds. GC-MS as analysing the most of the compounds. GC-MS as

an analytical method of option is its increased an analytical method of option is its increased

sensitivity & reliability even in very small sensitivity & reliability even in very small

quantities (ng).quantities (ng).


M.Mcnair,M.Miller, basic gas chromatography- 2nd

Edition. (Pg.No 156-169)

David G.Watson, Pharmaceutical analysis. (Pg.No

202-206).

AH Beckett, J.B Stenlake, Pharmaceutical

chemistry 4th Edition-Part two (Pg.No 474-477).

Skoog, Holler, Lrouch, Instrumental-Analysis.

(Pg.No 606-629).


Gurdeep R.Chatwal, K.Anand, Instrumental

methods of chemical analysis (Pg.No

2.272,2.673)

Pavia,Lampman, Kriz, (Pg.No 401-415).

B.K Sharma, Instrumental methods of chemical

analysis (Pg.No 844-938,180-224)

Dr.S.Ravi Sankar text book of pharmaceutical

analysis 3rd Edition (Pg.No 8.1,17.1)



ACKNOWLEDGEMENTACKNOWLEDGEMENTS

I sincerely thank my guide

Mr. Ch. DEVADASU sir

for his constant guidance &

support.

I thank our respected Principal

Dr. P.SRINIVASA BABU

& seminar Committee for giving me

this opportunity.


Education

GCMS