Hyphenated Techniques - coupling of a separation technique and an on-line spectroscopic detection technology.
Advantages of hyphenated techniques;
1. Fast and accurate analysis.
2. Higher degree of automation.
3. Higher sample throughput.
4. Better reproducibility.
5. Reduction of contamination due to its closed system.
6. Separation and quantification achieved at same time.
1. Presented By :
Mr. Sanket Rajiv Shinde
M. Pharmacy (QAT)
Savitribai Phule Pune University
2. Contents
1. Introduction
2. List of Hyphenated Techniques
3. GC-MS
4. LC-MS
5. GC-IR
6. LC-ESI-MS
7. Advantages of Hyphenated Techniques
8. References
2
3. What Is Hyphenated Technique ?(1)
The term “hyphenation” was first adapted by Hirschfeld in
1980.
The technique developed from the coupling of a separation
technique and an on-line spectroscopic detection technology
is known as hyphenated technique.
3
Introduction
7. Principles :
GC :
• May be Gas Liquid [GLC] or Gas Solid Chromatography [GSC]
but GLC is preferred
• GLC works by partition but GSC works by adsorption
• In GLC the substance to be studied first converted to gas which
works as the mobile phase.
MS :
1. Ion Formation
2. Ion Separation & Detection 7
GC-MS conti….
8. • Techniques used to form ions are:
1. Electron Ionisation
2. Chemical Ionisation
3. Desorption Ionisation
i. Secondary Ion Mass Spectroscopy [SIMS]
ii. Fast Atom Bombardment [FAB]
iii. Matrix Associated Laser Desorption
Ionisation [MALDI]
4. Electrospray Ionisation
8
1. Ion Formation
GC-MS conti….
9. 2. Ion Separation and Detection
• Types of instruments are used:
i. Direct Focusing Type:
a) Single Focusing
b) Double Focusing
ii. Quadruple Mass Analyser
iii. Quadruple Mass Storage
iv. Time of Flight Mass Analyser
9
GC-MS conti….
10. Instrumentation of GC :
A. Carrier Gas:
• Chemically inert
• Suitable for the detector use
• Amount of impurity should be less
• Easily available and should be cheap
• Non inflammable
Generally used carrier gases are
He, H, N, Ar etc.
10
GC-MS conti….
11. B. Flow Regulators:
Used to maintain the
uniform pressure and
flow rate.
Generally used are:
• Rotameter
• Soap Bubble Flow Meter
11
GC-MS conti….
12. C. Injection Devices:
• GAS : Valve Devices
• LIQUID : Loop Devices
• SOLID : Dissolve the
substance in suitable
solvent and then inject.
12
GC-MS conti….
13. D. Columns:
Columns can be of the following types:
• Packed Columns [Stationary Phase- PEG]
• Open Tubular Column or Open Capillary Column/ GOLAY Column
• SCOT ( Support Coated Open Tubular Column)
13
GC-MS conti….
14. E. Temperature Controlling Devices:
The devices are very important role playing.
Generally used Temperature Controlling Devices are:
• Pre heaters(for vapour formation)
• Thermostatically controlled oven
14
GC-MS conti….
16. Principle of working and interfaces :
GC-MS works by :
1. Iso Thermal Principle
2. Liner Principle
Interfaces of GC MS are :
1. Molecular Separator
2. Permeation Separator
3. Open Split
4. Capillary Direct
16
GC-MS conti….
18. Gather Evidence
18
Police Officer
1. Interview witness
2. Dust for fingerprints
Mass Spectrometrist
1. Interview biologists who
isolated the protein
2. Cleave protein to obtain
peptide mixture
3. Analyse peptide mixture by MS
to obtain Peptide molecular
masses !
GC-MS conti….
19. 19
Database Search Results
Police Officer
Identifies the robber
Mr. XYZ
Mass Spectrometrist
Identifies the protein
e.g. bovine carbonic
anhydrase
GC-MS conti….
20. Applications of GC-MS :(1)
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.
20
GC-MS conti….
21. Sports antidoping analysis (in forensic GC-MS).
Analyzer of aerosol particles.
Determination of pesticide residues in food.
Polymer characterization (pyrolysis methods combined
GCMS).
Drug monitoring & toxicology studies.
Explosive analysis
Environmental analysis
21
GC-MS conti….
35. Detectors :
Records either the charge induced or the current produced
when an ion passes by or hits a surface.
A continuous dynode particle
multiplier detector
35
LC-MS conti….
39. Pharmacokinetics: Bio analysis
Drug development
Molecular weight determination
Toxicology
Impurity detection
Determination of isotopes
Proteomics/Metabolomics
Determination of pesticides, herbicides & organic
pollutants for environmental ,monitoring.
Applications of LC-MS :(1)
39
LC-MS conti….
40. Comparison of LC-MS with GC-MS :
• GC-MS requires compounds to be volatile to be ionised.
- traditionally electron impact source is used.
• LC-MS can be used to detect compounds from poly-
aromatic (non-polar) to peptide & proteins.
• GC-MS is still able to detect long chain aliphatic
compounds (petroleum based analytes) & very low mass
volatile material better than LC-MS.
40
41. Very sensitive, very expensive, sample recovery is also
possible because IR is non-destructive technique.
In this, GC does the separation part where as IR perform
function of identification.
Effluent from GC is directly forwarded into the heated
pipe of IR at atmospheric pressure.
IR spectroscopy identifies the compound by identifying
the functional group. 41
GC-IR
Gas
Chromatography
Infrared
Spectroscopy
GC-IR
42. 42
Applications of GC-IR :
DNA analysis of blood samples, other fluids.
Pharmaceutical applications
Industrial applications
GC-IR conti….
45. 1. Fast and accurate analysis.
2. Higher degree of automation.
3. Higher sample throughput.
4. Better reproducibility.
5. Reduction of contamination due to its closed system.
6. Separation and quantification achieved at same time.
45
Advantages of
Hyphenated Techniques:
46. 1. Kalpesh N Patel, et al ; Introduction to hyphenated techniques and their
applications in Pharmacy; Pharmaceutical Methods; Oct-Dec 2010; Vol 1 ;
Issue 1.
2. Silverstein R; Spectroscopic Identification of Organic Compounds; Wiley
Publication Delhi; 6th edition; 2009; Page 2-70
3. Skoog D et al; Fundamentals of Analytical Chemistry; Cengage Brain
Publication London; 9th edition; 2010; Page 16-25.
4. AH Beckett, J.B Stenlake, Pharmaceutical chemistry;4th edition-Part
2;Page 474-477.
5. Gurdeep R. Chatwal, K. Anand; Instrumental methods of chemical
analysis; Page 2.272,2.673
6. G. C. Stafford et al.; International Journal of Mass Spectrometry and Ion
Processes, 60; 1984, Page 85 and Analytical Chemistry, 59; 1987, Page
1677.
7. https://www.jic.ac.uk/services/metabolomics/topics/lcms/why.htm
46
References