CM4106

Separation Methods
Gas Chromatography: Applications.
Hyphenated Techniques
Dr. Amalia Muoz
Fundacin CEAM. Euphore Laboratories
amalia@ceam.es
 Gas Chromatography
Qualitative Analysis

The characteristic parameter

for the identification of a Time elapsed
compound is the between the
RETENTION TIME (RT) dead point and the
or the peak maximum
CORRECTED RETENTION
TIME (RT)

RT and RT depend on:

Temperature
Stationary phase (column)
Carrier gas
Time elapsed between the
Flow injection point and the peak maximum.
Etc. Each solute has a characteristic retention time.

Small fluctuations could derived in wrong identification

It is not correct absolute RT data from literature with those obtained experimentally.
 Gas Chromatography
Qualitative Analysis

Relative Retention times

It is the ratio between the corrected the retention time of the analyte to identify
(RTa) and a substance used as a reference (RTref)

'
RT
RTr = a
'
RT ref

To obtain the RTr the reference compound must be added to the sample and to
the standards

The RT of the reference compound should be closed to the analytes.

 Gas Chromatography
Qualitative Analysis

Other method

The sample is separated into two aliquots.

The first aliquot is injected directly

A determined amount of a standard is added into the second aliquot and then
injected.

If we see a new peak in the second chromatogram, we can ensure that this
substance is not in the sample

while a signal of some of the peaks appear more intense in the second
chromatogram that substance exists in the sample
 Gas Chromatography
Qualitative Analysis

It could be simpler

Using selective detectors: ECD, NPD, PID, etc.

Using hyphenated techniques: GC-MS, GC-FTIR, GC-NMR

Using two or more detectors

 Gas Chromatography
Qualitative Analysis

Two Detectors

A single injection on two GC detectors yields two sets of data in one half of the time

Detector A Detector A

Injector Injector

Detector B Detector B

Parallel Dual Detector Configuration Post Column Split Configuration

Inyector Detector A Detector B

Series Configuration
(Detector A must be non-destructive)
 Gas Chromatography
Quantitative Analysis
3 important stages in a GC analysis,

1. The preparation of the sample.

2. The development of the separation and the production of the chromatogram

3. The processing of the data and the presentation of the results.

Each stage is equally important and if not carried out correctly the results will
be neither precise nor accurate.

Sample preparation can be

Simple: involving no more that diluting a known weight of sample with
mobile phase
Much more complex: including an extraction procedure followed by
derivatization and then dilution.

For some samples the preparation can be the most time consuming and
difficult part of the whole analysis.
 Gas Chromatography
Quantitative Analysis

Chromatogram

The response must be linear

Concentration
Mass

The response factor of each

compound is different
for each compound

Parameters that can be used:

Peak Height
Peak Area
 Gas Chromatography
Quantitative Analysis

Chromatogram

Area Normalization
The sum of the areas of all the peaks
PeakAreag corresponds to 100% of the solutes separated.
%g = 100
areas Only true if:
All the compounds are eluted
Same sensitivity

As the compounds usually do not have the

same sensitivity a correction factor should be
PeakAreag f g
%g = 100 applied
(areai f i ) Calibration curve fg =
mass
area
 Gas Chromatography
Quantitative Analysis

Chromatogram Internal Standard

An internal standard is a compound, not present in the sample, that is added in a

constant amount to samples and calibration standards.

The peak of compound must not overlap with the peaks of the analytes.
SI Method
6
A rea com pound/ area

5
y = 0.9978x
4 R2 = 0.9991 Advantages: manual injection
y = 0.497x
SI

3
R2 = 0.999
2
Disadvantages:
1 To analyse great number of
0
analytes
0 2 4 6 8 10 12 To find a good IS
mass compound/ mass SI

compound A compound B Lineal (compound A) Lineal (compound B)

 Gas Chromatography
Quantitative Analysis

Chromatogram External Standard

ES Method
6

5
y = 1.9841x
Area compound

4 R2 = 0.9991
y = 0.9981x
3
R2 = 0.9993
2

0 1 2 3 4 5 6
mass compound

compound A compound B Lineal (compound A) Lineal (compound B)

Advantages: simpler than IS.

Disadvantages: Sample injection reproducibility
Preferable Automatic injection or sample valve
 Gas Chromatography
DERIVATIZATION

Derivatization is the process of chemically modifying a compound

to produce a new compound which has properties that are
suitable for analysis using a GC

WHY?

To permit analysis of compounds not directly amenable to

analysis due to, for example, inadequate volatility or stability

Improve chromatographic behavior or detectability.

Derivatization is a useful tool allowing the use of GC and GC/MS

to be done on samples that would otherwise not be possible in
various areas of chemistry such as medical, forensic, and environmental
 Gas Chromatography
DERIVATIZATION

Increases volatility (i.e. sugars):

Eliminates the presence of polar OH, NH, & SH groups
Derivatization targets O,S, N and P functional groups (with hydrogens
available

Increases detectability, I.e. steroids/ cholesterol

Increases stability
Enhances sensitivity for ECD (Electron Capture Detection). The introduction of
ECD detectable groups, such as halogenated acyl groups, allows detection of
previously undetectable compounds

in some cases: derivatization can also be used to decrease volatility to allow

analysis of very low molecular weight compounds, to minimize losses in
manipulation and to help separate sample peaks from solvent peak.
 Gas Chromatography
DERIVATIZATION
Comments
Silylation Readily volitizes the sample
-Used as the first step to further
derivatizations or as a method of
protection of certain active hydrogens.
-Reduces the polarity of amino,
Acylation
hydroxyl, and thiol groups and adds
halogenated functionalities.
-Reduces molecular polarity by
replacing active hydrogens with an
alkyl group.
- modify compounds with acidic
Alkylation
hydrogens, such as carboxylic acids
and phenols.
-Reagents containing fluorinated
benzoyl groups can be used for ECD
Formation of
Reagents containing fluorinated
perfluoro-
benzoyl groups can be used for ECD
derivatives
GC-Quiral
Derivatiz.
Advantages Disadvantages
-Moisture sensitive
- Wide variety of compounds
-Organic solvents must be aprotic
-Large number of silylating reagents
(no protons available)
available
-WAX type columns cannot be
-Easily prepared
used
-Increased detectability by ECD
-Difficult to prepare.
-Derivatives are hydrolytically stable
- Reaction products often need to
- Increased sensitivity by adding molecular
be removed before analysis
weight
- Moisture sensitive
-Acylation can be used as a first step to
- Reagents are hazardous and
activate carboxylic acids prior to
odorous
esterfication (alkylation)
-Wide range of alkylation reagents.
-Reaction conditions can vary from -Limited to amines and acidic
strongly acidic to strongly basic hydroxyls
- Some reactions can be done in aqueous - Reaction conditions are
solutions frequently severe
- Alkylation derivatives are generally - Reagents are often toxic
stable
Wide range of application
Easy to prepare
Selectivity
 Gas Chromatography
DERIVATIZATION
Some examples

Derivatization Reaction Common Derivatizing Agent

Methylation of carboxylic acids Diazomethane,

methanol/sulfuric acid

Oxime formation of carbonyl functionality PFBHA

N-hexyl carbonate, carbamate, and ester formation N-hexyl chloroformate

from hydroxylic, aminic, and carboxylic functionality

Heptafluorobutyramide formation from aromatic amines Heptafluorobutyramide

And much more

 HYPHENATED TECHNIQUES in Chromatography

On-line combination of a chromatographic separation technique with a

sensitive and Element-specific detector

Chromatographic Techniques Spectroscopic Techniques

+ - + -
Separation of Necessity of
analytes pure analytes

Low security in High identification

identification level

Hyphenated techniques provide the analyst with structural

information on the components present in complex mixtures.

This information may be sufficient to identify components

 HYPHENATED TECHNIQUES in Chromatography

Column Interface Spectrometer

Chromatograph

Column
Non-Destructive
Detector
Interface Spectrometer
Chromatograph

Detector
Non-Destructive
Column Interface Spectrometer
Chromatograph

Interface 1 Non-Destructive
Column
Spectrometer
Chromatograph
Interface 2 Spectrometer

Interface 1 Spectrometer
Column
Chromatograph Interface 2 Spectrometer
 HYPHENATED TECHNIQUES in Chromatography
Common hyphenated techniques

Mass Spectrometry LC

S
S -M

IR-M
C - M S
G

-FT
GC
Infrared Spectroscopy LC-F
TIR
TIR
GC-F
Gas Liquid
Chromatography Chromatography
GC- Emission and Absorption ICP
AAS LC -
Atomic Spectroscopy

MR
C -N
Nuclear Resonance L
Spectrometry
 HYPHENATED TECHNIQUES in Chromatography
GC/MS
Mass Spectrometer

Provide Information about the chemical composition of the analyte

Molecular analysis
Isotopic analysis
Trace analysis
Elemental analysis
Surface analysis

High Detection efficiency and specificity of molecular recognition

Molecules are ionized (broken down) into electrically charged particles called
ions with a specific mass and charge.

Due to that, the speed and direction of the ion may be changed with an electric
or magnetic field.
HYPHENATED TECHNIQUES in Chromatography
GC/MS
 HYPHENATED TECHNIQUES in Chromatography
GC/MS
Mass Spectrometer
Components

GC Column/MS Interface

Ionizations Source: for example Electron Impact (EI) or Chemical Ionization (CI)

Mass Analyzer: for example Magnetic Sector, Quadrupole, Ion Trap, Time of Flight

Mass Detector

Software/Data display
 HYPHENATED TECHNIQUES in Chromatography
GC/MS
Mass Spectrometer
Ionization methods

Should provide a high ionization efficiency and high stability with minimum
kinetic energy distribution and minimum angular dispersion of the ion
produced

The ion source should produce:

Intact molecular ions MW (for MW information)

Fragment ions (for structural information)
Control over the internal energy transferred to the molecule for
control over the degree of fragmentation
Should be possible to couple with various types of chromatographs
HYPHENATED TECHNIQUES in Chromatography
GC/MS
Mass Spectrometer
Ionization methods
Electron Impact, 70 eV

Analytes elute here

A+ B+ C+ ABC
 HYPHENATED TECHNIQUES in Chromatography
GC/MS
Mass Spectrometer
Ionization Type of Ionization
Pressure Characteristics Application
method analysis agent
Electron Electrons Reproducible spectra,
Impact Molecular (70 eV) 10-5 Torr extensive fragmentation VOC; structural elucidation
(EI)

Chemical Gaseous ions. Molecular ions and

Ionization Molecular (Reagent gas: 1 Torr controllable VOC; MW determination
(CI) CH4, NH3, fragmentation
NF3, N2O)

Desorption Energetic 10-6-10-5 Intact molecular ions Condensed phase, high-mass

Ionization Molecular particles, Torr from high-mass compounds; MW and structure
(DI) photons compounds determination

Spray Electrical, Intact molecular ions Solutions of high-MW

Ionization Molecular thermal, and 1- 760 from high-mass compounds; used with LC/MS
(SI) pneumatic Torr compounds to determine MWs and
energy structures

Glow-
Discharge Elemental Plasma 0.1-10 Very stable, Elemental analysis of solids
Ionization Torr reproducible spectra
(GD)

Inductively Atmosp. High ionization

Coupled Elemental Plasma pressure efficiency Elemental analysis of solutions
Plasma (ICP)
HYPHENATED TECHNIQUES in Chromatography
GC/MS
Mass Spectrometer
Mass Analyzer

Function: to measure the mass-to-

charge ratios of ions (m/z), thus
providing a mesa to identify them.

Depend on the interactions of charged

particles with electric or magnetic
fields
HYPHENATED TECHNIQUES in Chromatography
GC/MS
Mass Spectrometer
Mass Analyzer

Quadrupole:

Orthogonal DC and RF planes (x/y) are used to separate

ion passing through a highly evacuated tube.

As an analyte mass fragment pass trough the mass

analyzer, the matched x/y field at that instant a very small
part of one scan- allows only specific fragment exit into the
mass detector. The duration of an entire scan is usually
less than 1 second (e.g. 0.6 sec). The m/z range can be
adjusted by the analyst

MS scanning process occurs very quickly, so each scan

includes a measure of all the amounts of each different
mass fragment during that scan
 HYPHENATED TECHNIQUES in Chromatography
GC/MS
Mass Spectrometer
Mass Analyzer
Ion trap

It is an three-dimensional analog of the quadrupole

mass filter.

Because forces operate in 3 directions, the electric fields can be used store ions in an
electrical bottle.
It consist of two end-cap electrodes of hyperbolic cross section that normally are operated
at ground potential. A rotationally symmetric electric quadrupole field is generated

At a given voltage, ions of a specific mass range are held oscillating in the trap. Initially,
the electron beam is used to produce ions and after a given time the beam is turned off.
All the ions, (except those selected by the magnitude of the applied rf voltage) are lost to
the walls of the trap, and the remainder, continue oscillating within the trap. The potential
of the applied rf voltage is then increased, and the ions sequentially assume unstable
trajectories and leave the trap via the aperture to the sensor. The ions exit the trap in
order of their increasing m/z values.
HYPHENATED TECHNIQUES in Chromatography
GC/MS
Mass Spectrometer
Mass Analyzer

Time-of-Flight:

A beam of ions is accelerated through a known potential V,

and the time taken to reach a detector at a distance d, in a
linear fligh tube, is measured.

If all ions fall through the same potential, V, their velocities

must be inversely proportional to the square roots of their
masses.

The source must be pulsed in order to avoid simultaneous

arrivals of ions of different mass-to-charge ratios

.
 HYPHENATED TECHNIQUES in Chromatography
GC/MS
Mass Spectrometer
Dynamic range
Resolution Mass (number of order
Mass/Charg at 1000 Measurement of magnitude of
Quantity Operating
Method e range (Da/charge) Accuracy at concentration
measured 1000 over which
Pressure
(Da/charge) (mass peak
witdh) Da/charge response varies
linearly)

Sector Momentum/
104 105 < 5 ppm 107 10-6
Magnet charge

Time of
Flight time 106 103 0.01% 104 10-6
flight

Quadrupole
frequency 104-105 103-104 0.1% 104 10-3
ion trap

Quadrupole Filters for m/z 103-104 103 0.1% 105 10-6

Cyclotron
frequency 105 106 < 10 ppm 104 10-9
Resonance
HYPHENATED TECHNIQUES in Chromatography
GC/MS
Mass Spectrometer
Mass Detector and data generation

One scan includes all the mass detector

current at each m/z ratio allowed to exit the
mass analyzer.

A chromatographic peak ( around10

seconds wide) can be scanned 16 or 17
times.

Each scan generates an individual mass

spectrum which is saved in the computers
hard drive
HYPHENATED TECHNIQUES in Chromatography
Coupling
GC/MS

Direct Introduction

Interfaces for GC/MS

Jet separator
Permselective membrane
Molecular effusion
Direct coupling

Interfaces for LC/MS

Total solvent elimination before ionization
Partial Solvent elimination before ionization
Solvent introduction
 HYPHENATED TECHNIQUES in Chromatography
Coupling
GC/MS

JET SEPARATOR The GC flow is introduced into an evacuated

chamber through a restricted capillary.

At the capillary tip a supersonic expanding jet of

analyte and carrier molecules is formed and its
core area sampled into the mass spectrometer.
In an expanding jet, high molecular mass
compounds are concentrated in the core flow
whereas the lighter and more diffusive carrier
molecules are dispersed away, in part through
collisions.

Thus, sampling of the core flow produces an

J. Abian. JOURNAL OF MASS
SPECTROMETRY
J. Mass Spectrom. 34, 157-168 (1999)
enrichment of the analyte.
The jet interface is very versatile, inert and
efficient, despite disadvantages of reduced
efficiency with more volatile compounds and
potential plugging problems at the capillary
restrictor.
 HYPHENATED TECHNIQUES in Chromatography
Coupling
GC/MS

PERMSELECTIVE MEMBRANE

It is made of a silicone-rubber membrane

that transmits organic non-polar molecules
and acts as a barrier for (non-organic)
carrier gases.

Despite being a very effective enrichment

procedure, it also suffers from
discrimination effects with more polar
analytes and produces significant band
broadening of their chromatographic peaks
 HYPHENATED TECHNIQUES in Chromatography
Coupling
GC/MS

MOLECULAR EFFUSION
It is based on the molecular filtering of the
gas effluent by means of a porous glass frit.

The column effluent passes through a fritted

tube situated in a vacuum chamber.

Small molecules traverse the microscopic

pores in the tube walls and are evacuated
whereas high molecular mass molecules
are transferred to the ion source.

Drawbacks : High dead volume added and

high surface area.

Shows discrimination effects in the

case of smaller molecules
 HYPHENATED TECHNIQUES in Chromatography
Coupling
GC/MS

DIRECT INTRODUCTION
Capillary columns use optimum flow-rates
of gas of about 1-2 ml min-1, instead of
more than 10-20 ml min-1 used with packed
columns, allowing all the effluent to be
directed to the mass spectrometer.

This is usually done through a direct

coupling where the column exit is
introduced into the ion source without a
capillary restriction.

Extensively used in analytical laboratories.

Enrichment interfaces have become unnecessary for GC/MS

 HYPHENATED TECHNIQUES in Chromatography
Coupling
LC/MS

DIRECT LIQUID INTRODUCTION (DLI)

Use of the solvent as the reagent gas in a CI source

A major problem in the introduction of liquids through a capillary

is that the high vacuum in the ion source produces rapid
evaporation of the liquid inside the capillary, eventually leading to
flow stoppage through freezing of the solvent.

The use of restricted capillaries and the heating of the capillary in

part solved this problem

Maximum flow-rates accepted by DLI interfaces are in the range 50-

100 l min-1 and are best suited for micro- and nanobore
chromatography (<1 mm i.d.column)

Obsolete
 HYPHENATED TECHNIQUES in Chromatography
Coupling
LC/MS
THERMOSPRAY
In the presence of a volatile buffer in the solvent, ions are obtained without any other ionizing source

The liquid flowing through the hot capillary is partially evaporated so that an ultrasonic spray of vapour and
charged microdroplets is obtained at the probe exit.

Ions present in the source are transferred into the mass spectrometer through the ion cone aperture while the
main portion of the residual vapours is captured by the high-conductance vacuum line and purged by a rotary
pump.

Charged microdroplets in the spray are the result of the rapid breakdown of the liquid surface during
vaporization and the statistical distribution of electrolyte ions in the droplets. Hence, an equal mixture of positive
and negatively charged droplets is formed from a neutral solution.

Gas-phase ions are produced in the source from these microdroplets as a result of several processes including
ion desolvation and ion evaporation from the charged microdroplets, as well as gas-phase CI processes

TO VACUUM
 HYPHENATED TECHNIQUES in Chromatography
Coupling
LC/MS
THERMOSPRAY
The evaporation of neutral molecules from the initial charged droplet produces a reduction in droplet size
and a charge density increment.
This excess of electrical energy can produce either fragmentation of the droplets into smaller droplets or
desorption of ions into the gas phase by ion evaporation.
Alternatively, gas-phase ions can be produced by simple desolvation of liquid-phase ions.
These processes produce a plasma of reagent ions mainly derived from the ammonium acetate buffer in the
solution.
If the proton affinity is relatively high, the analyte will keep its charge or will take it from the reagent plasma.
If the analyte proton affinity is low relative to other reagent plasma molecules, it will not be ionized or ion-
molecules adducts will be observed.

TSP ionization causes simple soft ionization spectra containing mostly molecular information

TO VACUUM
 HYPHENATED TECHNIQUES in Chromatography
Coupling
LC/MS
Atmospheric Pressure ionization

Ionization of the column effluent is carried out at atmospheric pressure by any of

several procedures including a radioactive source, electrical discharges and high
voltage electric fields.

The ions produced are continuously sampled through a small aperture and pass
into the spectrometer where they are mass analysed.

Introduce only a small amount of solvent into the low-pressure region of the MS.

Two main commercial source types:

Atmospheric Pressure Chemical Ionization (APCI)

Electrospray (ESI) source
 HYPHENATED TECHNIQUES in Chromatography
Coupling
LC/MS
APCI
An atmospheric pressure vaporization chamber in which 1-2 l of a liquid sample is injected
through a septum. A hot carrier gas (N2) is introduced into the chamber to help vaporization
and to transport the analyte to an area close to a radioactive beta source (63Ni).
In this area gas ionization occurred, producing an atmospheric pressure reagent plasma
that gave rise to analyte ions through ion- molecule reactions.
Ions a then transferred to the mass spectrometer through a small aperture and mass
analysed

Analysis of medium- and low-polarity compounds or when relatively non-polar solvents

have to be used.
 HYPHENATED TECHNIQUES in Chromatography
Coupling
LC/MS

ESI

The liquid sample is introduced into a high potential chamber through the capillary and at
the exit an electrically induced spray of charged microdroplets is produced.

Ions in these droplets enter the gas phase through evaporative processes (similar to TSP).
The ions in the spray are captured through a glass capillary restrictor where they are
conducted into the low vacuum area of the mass spectrometer.
To promote droplet desolvation, the ionization chamber is continuously supplied with a
countercurrent flow of dry nitrogen.