Upload
others
View
28
Download
0
Embed Size (px)
Citation preview
CM4106 Separation Methods
Gas Chromatography: Applications. Hyphenated Techniques
Dr. Amalia Muñoz
Fundación CEAM. Euphore Laboratories
Gas ChromatographyGas ChromatographyQualitative Analysis
Time elapsed between the injection point and the peak maximum.
Each solute has a characteristic retention time.
Time elapsed between the
dead point and the peak maximum
Small fluctuations could derived in wrong identificationIt is not correct “absolute” RT data from literature with those obtained experimentally.
The characteristic parameter for the identification of a compound is theRETENTION TIME (RT)or the CORRECTED RETENTION TIME (RT’)
RT and RT’ depend on:TemperatureStationary phase (column)Carrier gasFlowEtc.
Gas ChromatographyGas ChromatographyQualitative Analysis
Relative Retention times
It is the ratio between the corrected the retention time of the analyte to identify (RT’a ) and a substance used as a reference (RT’ref )
'
'
ref
ar RT
RTRT =
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 ChromatographyGas ChromatographyQualitative 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 ChromatographyGas ChromatographyQualitative 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 ChromatographyGas Chromatography
Two DetectorsTwo Detectors
A single injection on two GC detectors yields two sets of data in one half of the time
Detector A
Detector B
Injector
Parallel Dual Detector Configuration
Detector A
Detector B
Injector
Post Column Split Configuration
Qualitative Analysis
Series Configuration
Detector A Detector BInyector
(Detector A must be non-destructive)
Gas ChromatographyGas ChromatographyQuantitative 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 ChromatographyGas ChromatographyQuantitative Analysis
Chromatogram
The response must be linearConcentrationMass
The response factor of each compound is different for each compound
Parameters that can be used:
Peak HeightPeak Area
Gas ChromatographyGas ChromatographyQuantitative Analysis
Chromatogram
Area Normalization
100% ⋅∑
=areas
PeakAreag g
100)(
% ⋅⋅∑⋅
=ii
gg
fareafPeakArea
g
The sum of the areas of all the peaks corresponds to 100% of the solutes separated.
Only true if: All the compounds are elutedSame sensitivity
As the compounds usually do not have the same sensitivity a correction factor should be applied
Calibration curveareamassfg =
Gas ChromatographyGas ChromatographyQuantitative 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
y = 0.9978xR2 = 0.9991
y = 0.497xR2 = 0.999
0
1
2
3
4
5
6
0 2 4 6 8 10 12mass compound/ mass SI
Are
a co
mpo
und/
are
a SI
compound A compound B Lineal (compound A) Lineal (compound B)
Advantages: manual injection
Disadvantages: To analyse great number of analytesTo find a good IS
Gas ChromatographyGas ChromatographyQuantitative Analysis
Chromatogram External Standard
Advantages: simpler than IS. Disadvantages: Sample injection reproducibilityPreferable Automatic injection or sample valve
ES Method
y = 1.9841xR2 = 0.9991
y = 0.9981xR2 = 0.9993
0
1
2
3
4
5
6
0 1 2 3 4 5 6mass compound
Are
a co
mpo
und
compound A compound B Lineal (compound A) Lineal (compound B)
Gas ChromatographyGas Chromatography
DERIVATIZATION
Derivatization is the process of chemically modifying a compoundto produce a new compound which has properties that are
suitable for analysis using a GC
To permit analysis of compounds not directly amenable toanalysis 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/MSto be done on samples that would otherwise not be possible invarious areas of chemistry such as medical, forensic, and environmental
WHY?
Gas ChromatographyGas 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 ChromatographyGas Chromatography
DERIVATIZATIONComments Advantages Disadvantages
Silylation Readily volitizes the sample
- Wide variety of compounds-Large number of silylating reagents available-Easily prepared
-Moisture sensitive-Organic solvents must be aprotic (no protons available)-WAX type columns cannot be used
Acylation
-Used as the first step to further derivatizations or as a method of protection of certain active hydrogens. -Reduces the polarity of amino, hydroxyl, and thiol groups and adds halogenated functionalities.
-Increased detectability by ECD-Derivatives are hydrolytically stable- Increased sensitivity by adding molecular weight-Acylation can be used as a first step to activate carboxylic acids prior to esterfication (alkylation)
-Difficult to prepare.- Reaction products often need to be removed before analysis- Moisture sensitive- Reagents are hazardous and odorous
Alkylation
-Reduces molecular polarity by replacing active hydrogens with an alkyl group. - modify compounds with acidic hydrogens, such as carboxylic acids and phenols. -Reagents containing fluorinated benzoyl groups can be used for ECD
-Wide range of alkylation reagents. -Reaction conditions can vary from strongly acidic to strongly basic- Some reactions can be done in aqueous solutions- Alkylation derivatives are generally stable
-Limited to amines and acidic hydroxyls- Reaction conditions are frequently severe- Reagents are often toxic
Formation of perfluoro- derivatives
Reagents containing fluorinated benzoyl groups can be used for ECD
Wide range of applicationEasy to prepareSelectivity
GC-Quiral Derivatiz.
Gas ChromatographyGas Chromatography
DERIVATIZATION
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 from hydroxylic, aminic, and carboxylic functionality
N-hexyl chloroformate
Heptafluorobutyramide formation from aromatic amines Heptafluorobutyramide
Some examples
And much more…
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Chromatographic Techniques Spectroscopic Techniques
Separation of analytes
Necessity of pure analytes
Low security in identification
High 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
On-line combination of a chromatographic separation technique with a sensitive and Element-specific detector
ChromatographColumn Interface Spectrometer
Chromatograph
Non-DestructiveDetector Interface Spectrometer
Column
Chromatograph
DetectorNon-Destructive
SpectrometerColumn Interface
Chromatograph
Non-DestructiveSpectrometerColumn Interface 1
SpectrometerInterface 2
Chromatograph
SpectrometerColumn
Interface 1
SpectrometerInterface 2
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Gas Chromatography
Mass Spectrometry
Infrared Spectroscopy
Emission and Absorption Atomic Spectroscopy
Nuclear Resonance Spectrometry
Liquid Chromatography
GC-MSG
C-FT
IR-M
S
GC-FTIR
GC-AAS
LC-MS
LC-FTIR
LC-ICP
LC-NMR
Common hyphenated techniques
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
GC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Provide Information about the chemical composition of the analyte
Molecular analysisIsotopic analysisTrace analysisElemental analysisSurface analysis
Mass SpectrometerMass Spectrometer
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.
GC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
GC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Mass SpectrometerMass SpectrometerComponents
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
GC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Mass SpectrometerMass SpectrometerIonization 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
GC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Mass SpectrometerMass SpectrometerIonization methods
ABC
Analytes elute here
Electron Impact, 70 eV
A+ B+ C+
GC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Mass SpectrometerMass SpectrometerIonization Ionization
methodmethodType of analysis
Ionization agent Pressure Characteristics Application
Electron Impact
(EI)Molecular Electrons
(∼70 eV) ∼10-5 Torr Reproducible spectra, extensive fragmentation VOC; structural elucidation
Chemical Ionization
(CI)Molecular
Gaseous ions. (Reagent gas:
CH4 , NH3 , NF3 , N2O)
∼
1 TorrMolecular ions and
controllable fragmentation
VOC; MW determination
Desorption Ionization
(DI)Molecular
Energetic particles, photons
10-6-10-5
TorrIntact molecular ions
from high-mass compounds
Condensed phase, high-mass compounds; MW and structure
determination
Spray Ionization
(SI)Molecular
Electrical, thermal, and pneumatic
energy
1- 760 Torr
Intact molecular ions from high-mass
compounds
Solutions of high-MW compounds; used with LC/MS
to determine MW’s and structures
Glow- Discharge Ionization
(GD)Elemental Plasma 0.1-10
TorrVery stable,
reproducible spectra Elemental analysis of solids
Inductively Coupled
Plasma (ICP)Elemental Plasma Atmosp.
pressureHigh ionization
efficiency Elemental analysis of solutions
GC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Mass SpectrometerMass SpectrometerMass 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
GC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Mass SpectrometerMass SpectrometerMass 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
GC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Mass SpectrometerMass SpectrometerMass Analyzer
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.
Ion trap
It is an three-dimensional analog of the quadrupole mass filter.
GC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Mass SpectrometerMass SpectrometerMass 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
.
GC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Mass SpectrometerMass Spectrometer
MethodMethod Quantity measured
Mass/Charg e range
(Da/charge)
Resolution at 1000
(Da/charge)(mass peak
witdh)
Mass Measurement Accuracy at
1000Da/charge
Dynamic range (number of order of magnitude of concentration
over which response varies
linearly)
Operating Pressure
Sector Magnet
Momentum/charge 104 105 < 5 ppm 107 10-6
Time of flight Flight time 106 103 0.01% 104 10-6
Quadrupole ion trap frequency 104-105 103-104 0.1% 104 10-3
Quadrupole Filters for m/z 103-104 103 0.1% 105 10-6
Cyclotron Resonance frequency 105 106 < 10 ppm 104 10-9
GC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Mass SpectrometerMass SpectrometerMass 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 computer’s hard drive
CouplingGC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Direct Introduction
Interfaces for GC/MS
Jet separatorPermselective membraneMolecular effusionDirect coupling
Interfaces for LC/MSTotal solvent elimination before ionizationPartial Solvent elimination before ionizationSolvent introduction
CouplingGC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
The GC flow is introduced into an evacuatedchamber 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 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.
J. Abian. JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 34, 157-168 (1999)
JET SEPARATORJET SEPARATOR
CouplingGC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
It is made of a silicone-rubber membrane that transmits organic non-polar moleculesand 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
PERMSELECTIVE MEMBRANEPERMSELECTIVE MEMBRANE
CouplingGC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
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 microscopicpores in the tube walls and are evacuatedwhereas high molecular mass molecules are transferred to the ion source.
Drawbacks : High dead volume added andhigh surface area.
Shows discrimination effects in thecase of smaller molecules
MOLECULAR EFFUSIONMOLECULAR EFFUSION
CouplingGC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
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 packedcolumns, 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.
DIRECT INTRODUCTIONDIRECT INTRODUCTION
Enrichment interfaces have become unnecessary for GC/MSEnrichment interfaces have become unnecessary for GC/MS
CouplingLC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
DIRECT LIQUID INTRODUCTION (DLI)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 μl min-1 and are best suited for micro- and nanobore chromatography (<1 mm i.d.column)
Obsolete
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
CouplingLC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
THERMOSPRAYTHERMOSPRAYIn the presence of a volatile buffer in the solvent, ions are obtained without any other ionizing source
TO VACUUM
CouplingLC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
THERMOSPRAYTHERMOSPRAYThe 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
CouplingLC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
Atmospheric Pressure ionizationAtmospheric 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
CouplingLC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
APCIAPCIAn atmospheric pressure vaporization chamber in which 1-2 μl 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.
CouplingLC/MS
HYPHENATED TECHNIQUES in ChromatographyHYPHENATED TECHNIQUES in Chromatography
ESIESI
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.