HYPHENATED TECHNIQUES Gas chromatography- Mass spectrometry( GC-MS) and Gas chromatography- Atomic absorption spectroscopy( GC-AAS ) Hyphenated techniques. Advanced Instrumental analysis/ Pharmaceutical analysis

1. GAS CHROMATOGRAPHY - MASS SPECTROMETRY
(GC-MS) AND GAS CHROMATOGRAPHY - ATOMIC
ABSORPTION SPECTROSCOPY (GC-AAS)
. PRESENTED BY:
SAMIKSHA BOBDE
M.PHARM. I YEAR ( PHARMACEUTICAL CHEMISTRY )
DEPARTMENT OF PHARMACEUTICAL SCIENCES, RASHTRASANT
TUKADOJI MAHARAJ NAGPUR UNIVERSITY

2. INTRODUCTION – HYPHENATED TECHNIQUES
 It is define as the combination or hyphenation between Spectroscopic and separation
(chromatographic) technique is known as Hyphenated Technique.
 Spectroscopic + Chromatographic Hyphenation Hyphenated Technique.
 Hyphenated techniques combines chromatographic and spectral method to exploit the
advantages of both. Chromatography produces pure fractions of chemical components in a
mixture.
 Spectroscopy produces selective information for identification using standards or library
spectra. Mass spectrometer provides information that aids in the identification and structural
elucidation of each component.

3. GAS CHROMATOGRAPHY- MASS SPECTROMETRY
(GC-MS)
Gas
Chromatography
Separates mixture
of components into
individual
Mass
Spectrometry
GC-MS
Gas chromatography-
Mass spectrometry
Identifies (detects)
molecules based on
their molecular
weight or mass
A chemical analysis
technique combining two
instruments to provide for
powerful separation and
identification

4. GAS CHROMATOGRAPHY
 Gas Chromatography is an analytical technique that helps to separate and analyze a
mixture of organic vaporizable or volatile compound.
 It runs on the principle of partitioning of volatile samples with gaseous mobile phase
and liquid stationary phase.
 It is a process of separating components from the given crude drug by using a gaseous
mobile phase.

5. BASIC INSTRUMENTATION OF GAS
CHROMATOGRAPHY
 Carrier Gas
 Pressure regulator
 Flow controller
 Sample injection port
 Column
 Detectors
 Recorder

6.  Carrier Gas:
 Served as mobile phase.
 Supplied in the steel tank under high pressure of 40-80 psi this passes into flow controllers.
 Example-nitrogen, helium, hydrogen and argon can also be used.
 Carrier gas should be inert, suitable for detector, should give best column performance and
economic.
 Flow controller: Allows the operator to adjust the flow rate to desired operating level.
 Sample injection port: Separately heated to a temperature slightly above the column to
made the analytical sample vaporize rapidly before entering to column.
 Column: 1) Capillary column – Inner Diameter is 1mm preferred is 0.25 and 0.32mm.
Type- 1) Wall coated open tubular column.
2) Support coated open tubular column.
2) Packed column – Inner diameter 2- 4.6mm. Made up of stainless steel or glass.

7.  Detectors: Detector is a device that converts some physical property of the molecule
such as thermal conductivity.
 Mass flow rate detectors – Sensitive to the rate of flow of solute through the
detector.
1) Flame ionization detector
2) Thermionic specific detector
 Concentration sensitive detectors –
1) Thermal conductivity detector
2) Electron capturing detector
 Applications:-
 Gas chromatography is used for the separation and analysis of mixtures.
 Analysis of toxic compounds, solvents, hydrocarbons as well in forensic field.
 Pollution studies, environmental analysis.

8. MASS SPECTROSCOPY
Mass spectrometry is the analytical technique in which a mixture of ions is
separated according to mass to charge ratios.
Sample Volatile
form
Ionize Molecula
r ions
Fragmentation
Daughter
ions
Detector

9. INSTRUMENTATION
Instrumentation:
 Sample Inlet System
 lonization Chamber
 Mass Analyzer
 Detector
 Vacuum System and Read Out System

10. GC-MS
 GC-MS, which is a hyphenated technique developed from coupling of GC and MS, was the
first of its kind to become useful for research and development process.
 Mass spectra obtained by this hyphenated technique offer more structural information based
on the interpretation of fragmentations. The fragment ions with different relative abundances
can be compared with library spectra.
 Compounds that are adequately volatile, small, and stable in high temperature in GC
conditions can easily be analyzed by GC-MS.
 In GC-MS, a sample is injected into the injection port of GC device, vaporized, separated in
the GC MS detector, and recorded.

11. PRINCIPLE OF GC-MS
 The sample solution is injected into the GC inlet where it is vaporized and swept onto a
chromatographic column by the carrier gas.
 The sample flows through column and compounds are separated by virtue of their relative
interaction with the coating of the column and the carrier gas.
 Latter part of column passes through a heated transfer line and ends at entrance to ion source
where compounds eluting from the column are converted to ions and detected according to
their mass to charge m/z ratio.

12. NEED AND ADVANTAGES 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 used for separation and identification of volatile compounds and 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.
 It is having high resolution and separation capacity.
 Good accuracy and precision.

13. INSTRUMENTATION OF GC-MS

14. Gas
Chromatography
• Carrier gas
• Flow controller
• Injector
• Column
• Oven
Interface
• Jet interface
• Direct capillary
infusion interface
Mass spectrometer
• Ion source
• High-vaccum
system
• Mass analyzer
• Ion collector
Data
system

15. APPLICATIONS OF GC-MS
 Structure and hydrocarbon analysis.
 Analysis of biologically important aromatic amines.
 Forensic (explosives, drugs, unknown).
 Environmental analysis pesticide analysis, food safety and quality.
 Pharmaceutical and drug analysis.
 Clinical toxicology.
 Food and fragrance.

16. GAS CHROMATOGRAPHY- ATOMIC
ABSORPTION SPECTROSCOPY (GC-AAS)
 This technique is combination of gas chromatography with atomic absorption spectroscopy.
 AAS is one of the elemental analysis techniques.
 GC performs the separation of the components and with the help of AAS the elemental
identification of the component is performed. Elemental composition of every peak separated
by GC is determined.
 GC effluent is directly introduced into the Quartz atomization furnace, analytes are first
atomized using microwave irradiation, where the atoms are transferred to electronically
excited state.
 Then these electrons are return to the lower energy levels at that time photons are emitted at
certain wavelength that are characteristic of the particular element. In both the techniques
sample is in gas phase so the techniques are complementary to each other.

17. ATOMIC ABSORPTION SPECTROSCOPY
Liquid
sample
Formation
of
droplets
Fine
residue
Formation of
neutral
atoms
Absorption of
radiation (provided
by source hollow
cathode lamp) of
specific wavelength by
neutral atoms
Measurement of
wavelength and
intensity of
absorbed radiation

18. Instrumentation:
 GC Unit
 Light source
 Atomizer
 Monochromators
 Detectors

19. INSTRUMENTATION OF GC-AAS
 GC Unit ( Gas Chromatography )
 Radiation source of AAS- 1) Hollow cathode lamp
2) Electrodeless discharge lamp
 Atomizers- Atomization is separation of particles into individual molecules and breaking
molecules into atoms. This is done by exposing the analyte to high temperatures in a flame or
graphite furnace atomizer converts the liquid into small droplets which are easily vapourised.
1) Flame atomisers –
 Total consumption burner- The sample is atomized into the flame, the sample solution, the
fuel, and oxidizing gases are passed separate passages to meet at the opening of the base of
flame. Flame breaks the sample in liquid form into the droplets evaporates and burns
leaving the residue which is reduced to atoms.
 Premixed burner - It is most widely used because of uniformity in flame intensity

20. 2) Non-flame atomisers - (Electrothermal atomizer)
 Nebulization- Conversion of the liquid sample to a fine spray
 Desolvation- Solid atoms are mixed with the gaseous fuel.
 Volatilization- Solid atoms are converted to a vapor in the flame.
 Monochromators: Prism monochromator: - Quartz material is used for making prism, as
quartz is transparent over entire region.
 Grating monochromator: - It consists of a series of parallel straight lines cut into a plane
surface.
 Detector: Photomultiplier Tubes :- 1) Made of a glass vacuum tube Photocathode
2) Several dynodes 3) One anode.

21. APPLICATIONS
 In determining the levels of metabolites in body fluids like plasma, serum, urine.
 Analysis of foods like carbohydrates, proteins, lipids, vitamins, steroids, drug and pesticide
residues.
 Dairy product analysis like milk, butter-for detection of aldehydes, milk sugars, ketones and
fatty acids.
 Separation and identification of volatile materials, plastics, natural and synthetic polymers,
paints and microbiological samples.
 GC-AAS is main tool used in sports anti doping laboratories to test athletes urine samples for
prohibited performance enhancing drugs. Ex: anabolic steroids.
 Impurity profiling of pharmaceuticals.
 Model impurity profiling: GC-AAS trace of commercial sample of propranolol with
impurities

22. REFERENCES
 Thakur P, Thakur U, Kaushal P, Ankalgi AD, Kumar P, Kapoor A, Ashawat MS. A review on
gc-ms hyphenated technique. Asian Journal of Pharmaceutical Analysis. 2021;11(4):285-92.
 Chauhan A, Goyal MK, Chauhan P. GC-MS technique and its analytical applications in
science and technology. J. Anal. Bioanal. Tech. 2014 Nov 1;5(6):222.
 Guo X, Lankmayr E. Hyphenated techniques in gas chromatography. Advanced Gas
Chromatography: Progress in Agricultural, Biomedical and Industrial Applications. 2012 Mar
21:3.
 Patel KN, Patel JK, Patel MP, Rajput GC, Patel HA. Introduction to hyphenated techniques
and their applications in pharmacy. Pharmaceutical methods. 2010 Oct 1;1(1):2-13.

23. Thank you