cm4106 separation methods gas chromatography: applications ... · separation methods gas...

CM4106 Separation Methods

Gas Chromatography: Applications. Hyphenated Techniques

Dr. Amalia Muñoz

Fundación CEAM. Euphore Laboratories

[email protected]

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.


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.


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


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.


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


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 =


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


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)


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?


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.


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.


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


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


GC/MS


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


GC/MS


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


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


Mass SpectrometerMass SpectrometerIonization methods

ABC

Analytes elute here

Electron Impact, 70 eV

A+ B+ C+

GC/MS


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


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



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



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



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


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


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


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


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


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


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


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


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


THERMOSPRAYTHERMOSPRAYIn the presence of a volatile buffer in the solvent, ions are obtained without any other ionizing source

TO VACUUM

CouplingLC/MS


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


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


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


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.

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