DEFINITION OF A HYPHENATED

INSTRUMENT
TWO (OR MORE ) INSTRUMENTS AUTOMATED
TOGETHER AS A SINGLE INTEGRATED UNIT VIA
A HARDWARE INTERFACE WHOSE FUNCTION IS
TO RECONCILE THE (OFTEN EXTREMELY
CONTRADICTORY) OUTPUT LIMMITATIONS OF
ONE INSTRUMENT AND THE INPUT LIMITATIONS
OF THE OTHER
 WHY HYPHENATION
 IN MANY CASES THE REQUIRED SELECTIVITY AND/ OR
SENSITIVITY CAN NOT BE OBTAINED WITH A SIMPLE
COMBINATION OF:
SAMPLE CLEAN-UP

TRACE ENRICHMENT
SEPARATION
And
DETECTION

STEPS
USING
LIQUID CHROMATOGRAPHY

OR
CAPILLARY GAS CHROMATOGRAPHY
 FEATURES BENEFITS
AUTOMATIONS REDUCTION OF : LABOUR
ERRORS
TIME
COST
MINATURIZATION SMALLER SAMPLES
LESS SOLVENT
LOWDISPERSION(>SIGNAL)
INTEGRATION SIMPLICITY
REDUCTION OF STABILITY

OPERATIONAL STEPS ROBUSTNESS

PROTECTION AGAINST LILGHT,

OXYGEN, LOSS OF SAMPLE
 ADVANTAGES OF HYPHENATED
TECHNIQUES

ADVANTAGE OF HYPHENATED TECHNIQUES IS THAT THE

MAIN TASKS IS SIMPLE PREPARATION:

REMOVAL OF INTERFERENCES

ENRICHMENT OF THE ANALYTE

PHASE TRANSFER OF THE ANALYTE

CAN OFTEN BE COMBINED IN A SINGLE STEP

 GAS CHROMATOGRAPHY-
MASS SPECTROSCOPY (GC-MS)
 INTRODUCTION
Like a good marriage GC and MS bring something to their union.
GC can separate volatile and semi- volatile components with great
resolution but it can not identify them. MS can provide detailed
structural information for identification of most compounds but it
can not readily separate them. Therefore the combination of these
techniques was suggested shortly after the development of GC in
mid 1950s.
GC and MS are highly compatible techniques. In both techniques
the samples are in vapour phase and both techniques deal with about
the same amount of the sample (less than 1 ng). Unfortunately there
is a major incompatibility between the two techniques.
The compound existing in the GC is a trace components in its
carrier gas at a pressure of 760 torr. But the MS operates at a
vacuum of about 10-6 to 10-5torr. This difference in pressure of 8 to
9 orders of a magnitude is a considerable problem
 SAMPLES
State
Organic compounds must be in solution for injection into the gas
chromatography. The solvent must be volatile and organic( for example,
hexane or dichloromethane).
Amount
Depending on the ionization method analytical sensitivity of 1 to
100pg/component.
Preparation
Ranges from simple dissolution of the sample in a suitable solvent to
extensive clean up procedures using various forms of LC
Analysis time
In addition to sample preparation time, the instrumental analysis time is fixed
by the duration of the gas chromatographic run, typically between 20 to 100
minutes. Data analysis can be 1 to 20 hours or more depending on the level
details necessary
GENERAL RULES
 Identification and quantification of volatile and semi
volatile organic compounds in complex mixtures
 determination of molecular weights and (sometimes)
elemental compositions of unknown organic
compounds in complex mixtures.
 structural determination of unknown organic
compounds in complex mixtures both by matching
their spectra with reference spectra and by priori
spectral interpretation.
 COMMON APPLICATIONS
 Quantification of pollutants in drinking water and
waste water using official U.S. Environmental
Protection Agency (EPA) methods.
 Quantification of drugs and their metabolites in blood
and urine for both pharmacological and forensic
applications
 Identification of unknown organic compounds in
hazardous waste dumps.
 Identification of reaction products by synthetic organic
chemists.
 Analysis of industrial products for quality control.
 Interfaces
 Gas Splitters
 The pressure incompatibility between GC and MS was solved in several
ways.The earliest approach dating from the late 1950’s simply split a small
fraction of the GC effluent into MS.Depending upon the pumping speed of the
MS about 1-5% of the GC effluent was split of into the MS,venting the
remaining 90-99% of the analyte into the atmosphere . However it was soon
realized that this was not the best way to maintain high sensitivity of the two
techniques.
 Carrier Gas Separators
Improved GC-MS interfaces were later designed which
reduce the pressure of GC effluent from about 760
Torr to 10-4 Torr. But at the same time passed all the
analyte molecules from the GC into MS. These
interfaces were no longer just GC carrier gas splitters
but carrier splitters but carrier gas separators. They
separated the carrier gas from the organic analytes and
increased their concentration in the gas stream. The
most important commercial GC carrier gas separator is
the jet Separator.
JET SEPARATOR
 DISADVANTAGES
 Packed GC columns are almost infinite source os small particles
upstream of the jet separator. If one of the particle escapes from the
column it can lodged in the spray office and stop the gas flow out the
GC column into the MS. Part of this problem can be eliminated with a
filter between the GC column and jet separator. But some times it is
not a particle at all but Tar (pyralized GC stationary phase that
accumulated in the spray office over a time which clearly requires
maintenance.
 Currently the most common strategy which is ideally suited for
capillary GC columns is to pass all the carrier gas flow in to the ion
source of the MS. This works only GC gas flow is sufficiantly small
and pumping speed of the MS is high to handle the gas flow. For most
capillary columns the gas flow is 1 to 2 ml/min and the pumping
speed ia at least 300li.per seconds. The development of flexible fused
silica capillary column as made this routine. Infact the only time a jet
separator is now used is a few applications that require packed or thick
stationary phase GC column. (for example for permanent gas analysis)
Capillary Column
Interface Heating
GAS CHROMATOGRAPHY-MASS
SPECTROSCOPY (GC-MS)
 GCMS
 AutoSpec-Q-ionoptics
Components of MS
Electron ionization is most commonly used to produce ions
from the compounds separated by GC. Chemical ionization
may also be used. Quadrupole, ion-trap and time-off-flight
analyzer are generally used to separate ions in the MS. These
analyzers have rapid response time and relatively low cost.
Data system
The amount of data that can be produced during once GC-MS
experiment is over-whelming. In a typical GC-MS experiment
the mass spectrometer might be scanned every two seconds
during a GC run to manage the high data flow computers are
required. It is virtually impossible to purchase a GC-MS
system with out a powerful computer acting as a data system.
 Various data output from GC-MS

1) TIC (Total Ion Chromatogram) chromatogram

from GC-MS
2) Mass Spectrum
3) Library search result
4) MC
5) SIM chromatogram
6) Calibration curve
7) Quantitative result, etc
 Analytical Information
GC-MS is used both for Qualitative identification and for the Quantitative
measurement of the individual components in complex mixtures. There are
different data analysis strategies for these two applications.
Qualitative
There are three ways of examining GC-MS data.
1) The analyte can go through the GC and look at the mass spectra scanned at
each GC peak maximum. This has the advantage of being relatively quick but
the disadvantage of missing components of the mixture that are not fully
resolved by GC.
2) Look at each mass spectrum in turn, in a sense stacking of the mass spectra
one behind the other and examining them individually. This has the advantage
of completeness but disadvantage of tedious
3) Look at the intensity of one particular mass as a function of time. This
approach makes use of three dimensional nature of the GC-MS data. Two of
these dimensions are the mass VS intensity and the third dimension is the GC
retention time over which the mass spectral data is required. The X-axis
represents GC retention time, Y-axis represents intensity and Z-axis represents
the mass. This plot is called mass chromatogram.
 Quantitation
GC-MS can be used to measure the
concentration of one or more analytes in a
complex mixture. Quantitation can be based on
peak areas from mass chromatograms or from
selected ion monitoring.
MASS SPECTRUM
 Standard Tests for GC-MS performance
The EPA has published a series of test to evaluate the performance of GC-MS systems
1) Sensitivity
2) Detection limit
3) Saturation recovery test
4) Spectrum validation
5) System suitability
Routine check of sensitivity is important since remedial action for example
source cleaning should be taken when the sensitivity falls to 1/3 of normal
value.
Spectrum validation is valuable for quadrupole instruments, this tests ensures that
the instrument can generate the spectra that are compatible with major
data bases which contain a large number magnetic sector instrument
spectra.
system suitability test provide the basis for a comprehensive test which can be
carried out regularly.
Saturation recovery test measure the ability of the system to acquire the mass
spectrum of low concentration compound following elution of large
amount of another component of comparable molecular weight.
The detection limit gives based on the summed intensityof selected peaks in the
spectrum of the test compound compared with the same number of the most
intense back ground peaks.
 Cost
Although in principle GC-MS experiments can be performed on
magnetic sector instruments, in practice almost all GC-MS today is
done on quadrupole or ion trap instruments. These instrument are
relatively inexpensive and are simple to control by a computer. The
major factor influencing the cost of a quadrupole - or ion trap based
GC-MS system is the ionization methods available on the instrument
and the mass range of the mass spectrometer. Simple quadrupole or
ion trap instruments that use only electron impact ionization and
have a mass range of 20 to 700 cost about $50,000. those capable of
both positive and negative chemical ionization and with mass ranges
of 20 to 2000 cost about $200,000. operating costs include
instrument maintenance, GC carrier gases and columns, and spare
parts. In most laboratories, these costs are about 5% of the
instrument cost per year.
 List of Vendors
 The Finnigan Corp. (San Jose. CA) USA
 Micro mass UK (Manchester, UK)
 The Hewlett-Packard corp. (Palo Alto, CA)
 Varian Associates, Inc. (Walnut Creek, CA) USA
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