Metabolomics : SCBT 33312

Lecture 5

Hyphenated Techniques
for analysis of biological samples

G. Prabhakaran 1
 Hyphenated Techniques

Hirschfeld, 1980. The coupling of a separation technique and an on-line

spectroscopic detection technology is known as hyphenated technique.
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 Hyphenated Technique : GC-MS

Rapid identification and characterization of known and new biomolecules

directly from different sources without the necessity of isolation and
purification can be achieved by various modern hyphenated techniques . 3
Hyphenated Techniques

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 List of Hyphenated Techniques 1. GC-MS 2. LC-MS 3. LC-NMR 4. EC-MS
5. CE-MS 6. GC-IR 7. LC-MS-MS 8. LC-ESI-MALDI-TOF 9. GC-MS-MS
10. GC-NMR 11. GC-AES 12. ICP-MS 13. ICP-AAS 14. ICP-OES
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Photo Diode Array detector
Solid phase extraction (SPE)
Large volume injection (LVI)

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Analytical Techniques in Simultaneous Estimation

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Advantages of Hyphenated Techniques

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GC-MS an advance analytical instrumental technique that combines the physical separation
capability of GC with the mass analyzing capability of MS.

Gas chromatography coupled with mass spectrometry is a versatile tool to separate,

quantify and identify unknown substances. 
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 The GC-MS
A mixture of compounds is separated by
gas chromatography, then identified by
mass spectrometry.

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Schematic diagram of GC-MS

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Mass spectrometry is based upon the motion of a charged particle, called an ion, in
an electric or magnetic field.  The mass to charge ratio (m/z) of the ion effects this
motion. 
1. a sample is loaded onto the MS instrument, and

2. the components of the sample are ionized by one of a variety of methods (e.g.,
by impacting them with an electron beam), which results in the formation of
charged particles (ions)

3. directing the ions into an electric and / or magnetic field

4. computation of the mass-to-charge ratio of the particles based on the details of

motion of the ions as they transit through electromagnetic fields, and

5. detection of the ions, which in step 4 were sorted according to m/z.

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It is important to understand the different applications GC-MS
and LC-MS offer for two different types of chemists, 1) analytical
and 2) synthetic organic chemists.

Organic chemists attempt to create a desired chemical structure

by transforming functional groups and intentionally breaking or
creating bonds; in their resulting identification procedures they
already have a relatively good idea of the chemical structure.

To characterize the resulting product the chemist will use Infrared

Spectroscopy (IR) to observe functional groups, Mass
Spectrometry (MS) to obtain the compound’s molecular
weight, and Nuclear Magnetic Resonance (NMR)
spectroscopy to determine the molecular structure.
Information from all three techniques is used to conclusively
identify the metabolite / analyte or synthesized product.

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 Different Types of MS
• GC-MS - Gas Chromatography MS
– separates volatile compounds in gas column and ID’s by
mass

• LC-MS - Liquid Chromatography MS

– separates delicate compounds in HPLC column and ID’s
by mass

• MS-MS - Tandem Mass Spectrometry

– separates compound fragments by magnetic field and ID’s
by mass

• LC/LC-MS/MS-Tandem LC and Tandem MS

– Separates by HPLC, ID’s by mass and AA sequence

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 Why MS/MS
• Unknown Identification
• Potentially two compounds of interest
have the same mass ( and same retention
time)
• Quantitation improvements ( background
signal reduced)
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 Hyphenated analytical techniques for the
characterization of carotenoids and their metabolites

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 Monitoring breath of
patients under
Analysing anaesthesia
molecules in space
Detecting traces
of toxic chemicals
in contaminated
marine life

Identifying
Gain further info unknown
about structure compounds
and chemical
properties of
molecules
Determine the
Detecting banned
abundance of
substances such
each isotope of an
as steroids in
element
athletes
 Words of Wisdom:
It is as important to understand what a
technique CANNOT tell you as it is to
understand what it can tell you.