GC-MS

Department of Pharmacy (Pharmaceutics) | Sagar savale
1December 12, 2015
Gas Chromatography – Mass Spectroscopy
[GC-MS]
Mr. Sagar Kishor Savale
[Department of Pharmacy (Pharmaceutics)]
2015-016
avengersagar16@gmail.com
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.

2December 12, 2015
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

3December 12, 2015
4. Column
5. Detector
6. Recorder
Figure 1 Instrumentation of Gas Chromatography

4December 12, 2015
2.6 Block Diagram of Gas Chromatography

5December 12, 2015
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

6December 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.

7December 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

8December 12, 2015
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.

9December 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
Temp(degC)
Time (min)

10December 12, 2015
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).

11December 12, 2015
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

12December 12, 2015
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

13December 12, 2015

14December 12, 2015
3. Electron Capture Detector
 The detector consists of a cavity that contains two electrodes and a radiation source that
emits  - radiation (e.g.63
Ni, 3
H)
 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

15December 12, 2015
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

16December 12, 2015
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 10-7
to 10-5
of Hg bombarded with beam
of electrons using tungsten or rhenium filament. Molecules are broken up into cations or
fragments.

17December 12, 2015
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)

18December 12, 2015
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)

19December 12, 2015
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

20December 12, 2015
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

21December 12, 2015
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

22December 12, 2015
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.

23December 12, 2015
• 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.

GC-MS

Recomendados

Recomendados

Más contenido relacionado

La actualidad más candente

La actualidad más candente (20)

Destacado

Destacado (20)

Similar a GC-MS

Similar a GC-MS (20)

Más de Sagar Savale

Más de Sagar Savale (20)

Último

Último (20)

GC-MS