GC-MS

Department of Pharmacy (Pharmaceutics) | Sagar savale

1 December 12, 2015

Gas Chromatography – Mass Spectroscopy

[GC-MS]

Mr. Sagar Kishor Savale

[Department of Pharmacy (Pharmaceutics)]

2015-016

[email protected]

Hyphenated Technique

It is define as the combination or Hyphenation between Spectroscopic and

separation (chromatographic) Technique is known as Hyphenated Technique.

Spectroscopic + Chromatographic Hyphenation Hyphenated Technique

1. Introduction

1.1 Gas Chromatography-Mass Spectroscopy [GC-MS]

1. It is one of the type of Hyphenated technique.

2. It is a combination of gas chromatographic technique and spectroscopic technique.

3. It is having a high resolution capacity.

4. It is used has volatile and Non-volatile compounds.

5. It is used for qualitative and quantitative analysis.

1.2 Need of GC-MS

It is important type of technique is used for the separation of organic and in organic compounds

and it is having ability for separation high molecular weight hydrocarbons. It is important type

of technique is used for separation and identification of volatile compounds. It is important for

determination of fragmentation pattern of compounds. It is also important for determination of

protein, peptides, amino acid, nucleic acid, as well as naturally or biological compounds. It is

one of the powerful technique is used for qualitative and quantitative analysis.

mailto:[email protected]


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1.3 Advantages of GC-MS

1. It is important for identification of compound.

2. It can Provides sensitive response to most analytes.

3. It is important to provide information of particular or specific class of compound.

4. It can provide information of structure or different structure of compound.

5. It is having high resolution and separation capacity.

6. It is time saving technique, having a high resolution capacity.

7. It is important determination of molecular weight as well as fragmentation pattern of

compound.

8. Good Accuracy and Precision.

9. It is simple, rapid, reproducible technique.

1.5 Parts of GC-MS

Gas Chromatography Ionization Mass analyser Detector

2. Gas Chromatography

It is Column Chromatography.

It is also known as Liquid chromatography.

2.1 Principle – Partition and adsorption Chromatography.

2.3 Type - GSC (gas solid chromatography)

GLC (gas liquid chromatography

2.4 Aim – Detection of Volatile compounds.

2.5 Instrumentation

1. Solvent reservoir system / Mobile phase

2. Degasser

3. Pump


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4. Column

5. Detector

6. Recorder

Figure 1 Instrumentation of Gas Chromatography


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2.6 Block Diagram of Gas Chromatography


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1. Carrier Gas

Serves as a mobile phase supplied in steel tanks under high pressure.

At a pressure of 40 to 80 psi passes into flow controllers which allows the operator to

adjust flow rate to desired operating level (50 to 100 mL/min.)

Usually nitrogen and helium are used.

Occasionally hydrogen and argon are used.

Purity of gas is very important because it deposits its impurities in the column.

It should be inert in respect with the sample component,

Column packing material.

Low viscosity gases as H2 and helium allow higher flow rates, while high viscosity gas

as nitrogen useful in reducing longitudinal diffusion.

Disadvantage is that in GC the mobile phase is limited.

1. Hydrogen

better thermal conductivity

disadvantage: it reacts with unsaturated compounds & inflammable

2. Helium

excellent thermal conductivity

it is expensive

3. Nitrogen

reduced sensitivity

it is inexpensive

4. Requirements of a carrier gas

Inertness

Suitable for the detector

High purity


6 December 12, 2015

Easily available

Cheap

Should not cause the risk of fire

Should give best column performance

2. Sample injection port

It is a small chamber, usually separately heated to a temp. Slightly above that of column. In

this the analytical sample is made to vaporize rapidly before entering the column. Sample is

introduced into the flowing gas stream through a self-sealing rubber or silicon septum using a

microliter syringe. Sample may be injected into chamber directly on the beginning of the

column. Samples may be pure liquids, solids dissolved in liquid solvents or gases.


7 December 12, 2015

3. Columns

• Important part of GC

• Made up of glass or stainless steel

• Glass column- inert , highly fragile

3.1 Columns Can Be Classified

Depending on its use

1. Analytical column

1-1.5 meters length & 3-6 mm d.m

2. Preparative column

3-6 meters length, 6-9mm d.m

3.2 Types of column

1. Packed column: columns are available in a packed manner

S.P for GLC: polyethylene glycol, esters, amides, hydrocarbons, polysiloxanes…

2. Open tubular or capillary column or Golay column

Long capillary tubing 30-90 M in length

Uniform & narrow d.m of 0.025 - 0.075 cm

Made up of stainless steel & form of a coil

Disadvantage: more sample cannot loaded

3. SCOT columns (Support coated open tubular column

Improved version of Golay / Capillary columns, have small sample capacity

Made by depositing a micron size porous layer of supporting material on the inner wall

of the capillary column

Then coated with a thin film of liquid phase


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4. Equilibration of the column

Before introduction of the sample

Column is attached to instrument & desired flow rate by flow regulators

Set desired temp.

Conditioning is achieved by passing carrier gas for 24 hours.


9 December 12, 2015

4. Temperature Control Devices

Preheaters: convert sample into its Vapoure form, present along with injecting devices

Thermostatically controlled oven : Temperature maintenance in a column is highly

essential or efficient separation.

4.1 Two types of operations

Isothermal programming

Linear programming - this method is efficient for separation of complex mixtures

Isothermal

Gradient

0

40

80

120

160

200

240

0 10 20 30 40 50 60

Tem

p (

de

g C

)

Time (min)


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5. Detectors

5.1 The requirements of an ideal detector are

Applicability to wide range of samples

Rapidity

High sensitivity

Linearity

Response should be unaffected by temperature, flow rate…

Non destructive

Simple & inexpensive

5.2 Type of detector

1. Thermal Conductivity Detector (Katharometer, Hot Wire Detector)

Measures the changes of thermal conductivity due to the sample (mg). Sample can be

recovered.

When a separated compound elutes from the column, the thermal conductivity of the

mixture of carrier gas and compound gas is lowered. The filament in the sample column

becomes hotter than the control column.

The imbalance between control and sample filament temperature is measured by a

simple gadget and a signal is recorded.

Measures heat loss from a hot filament

Filament heated to const T

when only carrier gas flows heat loss to metal block is constant, filament T remains

constant.

when an analyte species flows past the filament generally thermal conductivity goes

down, T of filament will rise. (Resistance of the filament will rise).



1.1 Relative Thermal Conductivity

Compound Relative Thermal Conductivity

Carbon Tetrachloride 0.05

Benzene 0.11

Hexane 0.12

Argon 0.12

Methanol 0.13

Nitrogen 0.17

1.2 Advantages of Katharometer

Linearity is good

Applicable to most compounds

Non destructive

Simple & inexpensive



1.3 Disadvantages

Low sensitivity

Affected by fluctuations in temperature and flow rate

Biological samples cannot be analyzed

2. Flame Ionization Detector

2.1 Destructive detector

The effluent from the column is mixed with H & air, and ignited.

Organic compounds burning in the flame produce ions and electrons, which can

conduct electricity through the flame.

A large electrical potential is applied at the burner tip

The ions collected on collector or electrode and were recorded on recorder due to

electric current.

FIDs are mass sensitive rather than conc. sensitive

2.2 Advantages

µg quantities of the solute can be detected

Stable

Responds to most of the organic compounds

Linearity is excellent

2.3 DA destroy the sample





3. Electron Capture Detector

The detector consists of a cavity that contains two electrodes and a radiation source that

emits - radiation (e.g.63Ni, 3H)

The collision between electrons and the carrier gas (methane plus an inert gas) produces

a plasma containing electrons and positive ions.

If a compound is present that contains electronegative atoms, those electrons are

captured and negative ions are formed, and rate of electron collection decreases

The detector selective for compounds with atoms of high electron affinity.

This detector is frequently used in the analysis of chlorinated compounds

e.g. – pesticides, polychlorinated biphenyls



3.1 Advantage

Highly sensitive

3.2 Disadvantage

Used only for compounds with electron affinity

6. Recorders & Integrators

Record the baseline and all the peaks obtained

7. Integrators

Record the individual peaks with Rt, height….

8. Derivatisation of sample

Treat sample to improve the process of separation by column or detection by detector.

They are 2 types

1. Precolumn Derivatisation - Components are converted to volatile & thermo stable

derivative.

9. Conditions –

9.1 Pre column Derivatisation

Component ↓ volatile

Compounds are thermo labile

↓ tailing & improve separation

9.2 Post column Derivatisation

Improve response shown by detector

Components ionization / affinity towards electrons is increased

10. Pretreatment of solid support

To overcome tailing

Generally doing separation of non-polar components like esters, ethers…

11. Techniques

1. Use more polar liquid S.P

2. Increasing amt. of liquid phase

3. Pretreatment of solid support to remove active sites.

2.7 Applications of GC

Separation

Purification



Quantification

Qualitative and quantitative

3. Mass Spectroscopy

It is important for determination of molecular weight of compound as well as

fragmentation pattern of compound.

3.1 Basic Principle

Samples are ionized

Some fragmentation usually occurs

Sample components (and fragments) are separated based on mass-to-charge ratio

Output is a mass spectrum

3.2 Principle

Organic molecules in gaseous state under pressure of 10-7to 10-5 of Hg bombarded with beam

of electrons using tungsten or rhenium filament. Molecules are broken up into cations or

fragments.



3.3 Ion Source

Type Name and Acronym Ionizing Process

Gas Phase Electron Impact (EI) Exposure to electron

stream

Chemical Ionization (CI) Reagent gaseous ions

Field Ionization (FI) High potential electrode

Desorption Field Desorption (FD) High potential electrode

Electrospray Ionization (ESI) High electric field

Matrix-assisted desorption ionization

(MALDI)

Laser beam

Plasma Desorption (PD) Fission fragments from 252Cf

Fast Atom Bombardment (FAB) Energetic atomic beam

Secondary Ion Mass Spectrometry

(SIMS)

Energetic beam of ions

Thermo spray Ionization (TS)



3.4 Different Ionization Methods

1. Electron Impact (EI - Hard method)

Small molecules, 1-1000 Daltons, structure

2. Fast Atom Bombardment (FAB – Semi-hard)

Peptides, sugars, up to 6000 Daltons

3. Electrospray Ionization (ESI - Soft)

Peptides, proteins, up to 200,000 Daltons

3.5 Types of Mass Analyzers

1. Magnetic field deflection

1. Magnetic Field only (unit resolution) (Single Focusing)

2. Double-Focusing

(Electrostatic & magnetic field, High resolution)

2. Quadrupole Mass Spectrometry

1. Quadrupole mass filter

2. Quadrupole Ion storage (Ion Trap)

3. Time of Flight

4. FT-ICR (Ion-Cyclotron Resonance)

5. MS/MS (Tandem Mass spectrometry)



3.6 Different Mass Analyzers

1. Magnetic Sector Analyzer (MSA)

High resolution, exact mass, original MA

2. Quadrupole Analyzer (Q)

Low (1 amu) resolution, fast, cheap

3. Time-of-Flight Analyzer (TOF)

No upper m/z limit, high throughput

4. Ion Trap Mass Analyzer (QSTAR)

Good resolution, all-in-one mass analyzer

5. Ion Cyclotron Resonance (FT-ICR)

Highest resolution, exact mass, costly

3.7 Detectors for MS

Two Basic Types

1. Electron Multipliers

2. Faraday Cup

Time of Flight (TOF) and Fourier Transform Ion-Cyclotron Resonance (FTICR) instruments

can separate more than one m/e- ratio simultaneously

3. Multiple detectors are required in this case



3.8 Applications mass spectroscopy

Molecular weight determination

Determination of molecular formula

Identification of elemental position of atoms in the molecule

Analysis of natural products and high polymers

Quantitative analysis of mixtures

Isotope abundance measurement (To distinguish between cis and trans isomers)

To study free radicals and determination of bond strength.

Analysis of closely related compounds like hydrocarbons, petroleum products.

Used for trace analysis of elements in alloys and minerals.

GC-MS INSTRUMENT



4. GC-MS works by

1. iso Thermal Principle

2. Liner Principle

4.1 Interfaces of GC MS are

1. Molecular Separator

2. Permeation Separator

3. Open Split

4. Capillary Direct

4.2 Application

1. Analysis of Natural Products and Traditional Herbal Medicine

2. Identification of Metabolite

3. Bio analysis / Bioequivalence Studies

4. ADME (Absorption, Distribution, Metabolism, and Excretion) Screening

5. Dissolution Testing

6. Method Development / Validation

7. Forced Degradation Studies

8. Impurity Profiling

9. Manufacturing / QA / QC

10. Analysis of amino acid

11. Determination of pesticides

12. Fingerprint

13. Pharmacokinetic studies



14. Biotechnology

15. Clinical research

16. Biochemical analysis

17. Cosmetic analysis

18. Environmental Analysis

19. Forensic Pharmacy

20. Pharmaceutical industries

5. Reference

• Silverstein R; Spectroscopic Identification Of Organic Compounds; Wiley Publication

Delhi; 6th Edition; 2009; Page 2-70

• Skoog D et al; Fundamentals of Analytical Chemistry; Cengage Brain Publication

London; 9th Edition; 2010; Page 16-25

• Kemp W; Organic Spectroscopy; Palgrave Macmillan Limited London UK; 1991; Page

72-75

• mtweb.mtsu.edu/nchong/MS%20Ion%20Sources-Ryan-6200.ppt cited on 11.02.2014

• Willard, Merritt, Dean, Settle ‘Instrumental methods of analysis’Seventh edition, CBS

Publishers & Distributers New Delhi 110002.

• Ning Ma, Bi-Kui hang, Huan-De Li et. al . Journal of Clinica Chimica Acta. 1-2, 380,

2007, 100-105.

• Hiren N. Mistri, Arvind G. Jangid, Mallika Sanyal et.al. Journal of Chromatography

B, 1-2, 850, 2007, 318-326.



• Willard H.H, Lyne L.M, John A.D, Fran S, Instrumenal methods of analysis, CBS

publication, Ed 7th , p 608-610.

• Ardrey, R. E.; Ardrey, Robert (2003). Liquid chromatography-mass spectrometry: an

introduction. London: J. Wiley. ISBN 0-471-49801-7.

• McMaster, Marvin C. (2005). LC/MS: a practical user's guide. New York: John Wiley.

ISBN 0-471-65531-7

• Wilfried M.A. Niessen, Wilfried M. Niessen (2006). Liquid Chromatography-Mass

Spectrometry, Third Edition (Chromatographic Science). Boca Raton: CRC. ISBN 0-

8247-4082-3.

• Elementary organic spectroscopy by Y.R.Sharma fourth edition 2007,by Rajendhra

Ravindra printers page(280-291).

• Spectroscopy of organic compounds by P.S.Kalsi sixth edition, by New age

international Pvt. Ltd. Page(415-439).

• Gurdeep R.Chatwal Sham K. Anand Instrumental Methods Of Analysis, Fifth addition,

Himalaya publishing house Pvt . Ltd.

• Pharmaceutical analysis (INSTRUMENTAL METHODS) by Dr . A.V.Kasture, ninth

edition.

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GC-MS

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