Download - Gas chromatography mass spectrometry
Hina QaiserMS-1 1st Semester
Department Of BiotechnologyLahore College For Women University
GC-MS is an integrated composite analysis Instrument Combining GC which is excellent in its ability for separation
with mass spectrometry ideal in identification and elucidate structure of separated component .
Gas Chromatography
• It separat
es components
of sample
Interface• Combin
es both techniques by removi
ng pressur
e incompatibility proble
m betwee
n GC and MS
Mass spectrometry• Ionise
eluted component and
separate,
identify it
according to
its mass to charge ratio
Gas Chromatography -Mass Spectrometry
Introduction
Gas chromatography-mass spectroscopy (GC-MS) is a hyphenated analytical technique
exquisitely sensitive but also specific and reliable
GC can separate volatile and semi-volatile compounds with great resolution, but it cannot identify them.
MS provide detailed structural information on most compounds such that they can be exactly identified, but
can’t readily separate them.
3
Therefore, marriage of both instruments have been proposed shortly after the development of GC in the mid-
1950s.
we obtain both qualitative and quantitative information of our sample in a single run within the same instrument
Today computerized GC/MS instruments are widely used in environmental monitoring ,in the regulation of agriculture
and food safety , and in the discovery and production of medicine.
Continued......
Principle of GC-MS
Instrumental Layout
GC-MS comprise following major block
The Gas Chromatog
raph
GC-MS Instrument
The insides of the GC-MS, with the column of the gas chromatograph in the oven on the right.
Gas Chromatography
Gas chromatography leads to Separation of volatile organic compounds
Separation occurs as a result of unique equilibrium established between the solutes and
the stationary phase (the GC column)
An inert carrier gas carries the solutes through the column
Basic Components:
Carrier Gas
Gas Controls
The Injector
The ColumnTwo Groups:
Packed Column
Capillary Column
The Oven
The Detector (Mass Spectrometer)
Continued......
Sample Preparation State
• Organic compounds must be in solution for injection into the gas chromatograph.
• The solvent must be volatile and organic (for example, hexane or dichloromethane).
Amount
Depending on the ionization method, analytical sensitivities of 1 to 100 pg per component are routine.
Preparation
• Sample preparation can range from simply dissolving some of the sample in a suitable solvent to extensive.
Carrier Gas
Hydrogen:better thermal conductivity.it
reacts with unsaturated compounds & inflammable
Helium:excellent thermal conductivity it
is expensive
Nitrogen:reduced sensitivity. It is
inexpensive
Requirements of a carrier gasInertness
Suitable for the detector
High purity (Better than 99.995%Better than 99.9995% for Mass Spec).
Easily available
Cheap
Should not cause the risk of fire
Should give best column performance
Soap Bubble Meter
Similar to Rota meter & instead of a float, soap bubble formed
indicates the flow rate
Flow regulators / Flow metersDeliver the gas with uniform pressure/flow rate
Rota meterPlaced before column inlet
It has a glass tube with a float held on to a spring.
The level of the float is determined by the flow rate of
carrier gas
Flow meters
Injection Devices
A GC syringe penetrates a septum to inject sample into the vaporization camber
Instant vaporization of the sample, 280 C
Carrier gas transports the sample into the head of the column
Purge valve controls the fraction of sample that enters the column
Purpose of Injection
Deposit the sample into the column in the narrowest band possible
The shorter the band at the beginning of the chromatographic process - tall narrow peaks
Gives maximum resolution and sensitivity
Therefore type of injection method and operating conditions is critical in obtaining precise and accurate results
Split or SplitlessMost common method of Injection into Capillary Columns
Most commonly misunderstood also!
Same injector hardware is used for both techniques
Electronically controlled Solenoid changes Gas Flow to determine Injector function.
Split InjectionMechanism by which a portion of the injected solution is discarded.
Only a small portion (1/1000 - 1/20) of sample goes through the column
Used for concentrated samples (>0.1%)
Can be performed isothermally
Fast injection speed
Injector and septa contamination not usually noticed
Splitless InjectionMost of the sample goes through
the column (85-100%)
Used for dilute samples (<0.1%)
Injection speed slow
Should not be performed isothermally
Controlled by solenoid valve
Requires careful optimisation
Splitless (100:90) vs. Split (100:1)
Injector
Syringe
Injector
Syringe
Purge valveopen
Purge valveclosed
GC column GC column
HeHe
On Column Injection
All of the sample is transferred to the column
Needle is inserted directly into column or into insert directly above
column
Trace analysis
Thermally labile compounds e.g Pesticides, Drugs
High molecular weight
Material of ConstructionMetal (1957)
Glass (1959)
Fused
Silica
(1979)
Aluminium Clad (198
4)
Inert Metal
(1990)
Columns
Capillary Column CharacteristicsLength (10m
– 50m)
Internal Diameter (0.1mm - 0.53mm)
Liquid Stationary
Phase
Film Thickness (0.1um -
5um)
Polarity (Non-polar - Polar)
Stationary PhasesChoice of phase determines selectivity
Hundred of phases available
Many phases give same separation
Same phase may have multiple brand names
Stationary phase selection for capillary columns much simpler
Like dissolves like
Use polar phases for polar components
Use non-polar phases for non-polar components
Choosing a Column
Internal Diameter
Film Thickness
LengthPhase
Internal DiameterSmaller ID’s
Good resolution of early eluting compounds
Longer analysis times
Limited dynamic range
Larger ID’s
Have less resolution of early eluting compounds
Shorter analysis times
Sufficient resolution for complex mixtures
Greater dynamic range
Film ThicknessAmount of stationary phase coating
Affects retention and capacity
Thicker films increase retention and capacity
Standard capillary columns typically 0.25µm
0.53mm ID (Megabore) typically 1.0 - 1.5µm
The maximum amount that can be injected without significant peak distortion
Column capacity increases with :-film thickness
temperature
internal diameter
stationary phase selectivity
If exceeded, results in :-peak broadening
asymmetry
leaking
Column Capacity
Length effects
Increasing the column length increases the resolution
Doubling the column length increases resolution by the factor of 1.4 but also increases the analysis time
Long column are employed when sample contains large number of componnets
length L = 30 m is the most common column used for many analyses (drugs, pesticides, PAHs)
Interfacing GC with Spectroscopic Methods
Elutes from column collected as separate fractions after being detected - composition measured by Mass Spectrometry.
GC equipment can be directly interfaced with rapid-scan Mass Spectrometers.
The flow rate is usually small enough to feed directly into the ionization chamber of the Mass Spectrometer.
Packed columns use a jet separator, which removes the carrier gas for the analyte.
Increase momentum of heavier analyte molecules so that 50% or more go into the skimmer.
Lighter helium molecules are deflected by vacuum and pumped away.
Use to identify components present in natural and biological systems.
odor/flavor of foods – pollutants.
Jet SeparatorTwo capillary tubes aligned with a small space between them. (1 mm)
A vacuum is created between the two tubes using a pump.
The GC effluent enters the vacuum region, those molecules which continue in the same direction enter the second capillary tube and continue to the ion source.
The carrier gas molecules are more easily diverted from the linear path by collisions.
The analyte molecules are much larger and carry more momentum.
The surface of the separator must be inactive and a reasonably even temperature.
Prone to leaks
Basic Mass Spec.Theory
• Mass Spec. is a Microanalytical Technique used to obtain information regarding structure and Molecular weight of an analyte
• Destructive method i.e sample consumed during analysis
• In all cases some form of energy is transferred to analyte to cause ionisation
• In principle each Mass Spectrum is unique and can be used as a “fingerprint” to characterise the sample
• GC/MS is a combination technique that combines the separation ability of the GC with the Detection qualities of Mass Spec.
Basic GCMS Theory• Sample injected onto column via injector• GC then separates sample molecules• Effluent from GC passes through transfer line
into the Ion Trap/Ion source• Molecules then undergo electron /chemical
ionisation• Ions are then analysed according to their
mass to charge ratio• Ions are detected by electron multiplier which
produces a signal proportional to ions detected
• Electron multiplier passes the ion current signal to system electronics
• Signal is amplified
• Result is digitised
• Results can be further processed and displayed
Basic GCMS Theory
Types of Ionisation
Electron impact ionisation
Chemical Ionisation
Electron Ionisation
• Sample of interest vaporised into mass spec
• Energy sufficient for Ionisation and Fragmentation of analyte molecules is acquired by interaction with electrons from a hot Filament
• 70 eV is commonly used
• Source of electrons is a thin Rhenium wire heated electrically to a temp where it emits free electrons
Definition of Terms
Molecular ion
The ion obtained by the loss of an electron from the molecule
Base peak
The most intense peak in the MS, assigned 100% intensity
M+ Symbol often given to the molecular ion
Radical cation
+ve charged species with an odd number of electrons
Fragment ions
Lighter cations formed by the decomposition of the molecular ion.
These often correspond to stable carbcations.
Electron Ionisation
Chemical ionisation
• Used to confirm molecular weight
• Known as a “soft” ionisation technique
• Differs from EI in that molecules are ionised by interaction or collision with ions of a reagent gas rather that with electrons
• Common reagent gases used are Methane , Isobutane and Ammonia
• Reagent gas is pumped directly into ionisation chamber and electrons from Filament ionise the reagent gas
Six Processes Occur in Mass Spectrometer.
1) Electrons are fired at the gaseous molecules…these knock off other electrons from some of the molecules…
2) M + electron M+ + 2e-
3) Gaseous ions are accelerated by passing through an electric field. At this stage they can be traveling at up to 2 x 10^5 m/sec. (about 1/1000 the speed of light.
4) They then pass through an electrostatic analyzer, which selects the ions of kinetic energy within a narrow range by using an electric field.
5) The fast moving ions now pass through the poles of electromagnet, where they are deflected.
6) The deflected ions pass through a narrow slit and are collected on a metallic plate connected to an amplifier. For a given strength of magnetic field, only ions of a certain mass pass through the slit and hit the collector plate. As the ions hit the plate they cause a current to flow through the amplifier. The more ions there are , the larger the current.
• The equation governing the deflection of ions in the magnetic field is as follows:
• r=
Where r= radius of circular path in the magnetic field
m= mass of ion
V=accelerating voltage
e=electrical charge on the ion
B=strength of strength
• Accelerating voltage V is kept constant
• Radius of the curvature is prop to
• Inversely prop to B
• To obtain a mass spectrum, the current through the elctromagnet is changed at a steady rate.
• Causes the magnetic field B to change its strength & hence allows ions of different
Mass/Charge value to pass successively through the slit.
• Mass spectrum produced plotting (ion current) against (electromagnetic current), which is equivalent to (relative abundance) against
(mass/charge (m/e) ratio).
3 Main Ways In Which Mass Spectrometry Is Applied To Determine
The Structures Of Organic Compounds
There are three main ways in which mass spectrometry is applied to the determination of the structures of organic compounds.
1. By measuring the relative heights of the molecular ion (M) peak and the (M+1) peak we can determine the number of carbon atoms in a molecule.
2. By measuring the accurate mass of a molecular ion we can determine its molecular formula.
3. By identifying the fragments produced when an ion breaks up inside a mass spectrometer we can often piece together the structure of the parent molecule.
Interpretation of Mass Spectra
•The MS of a typical hydrocarbon, n-decane is shown above. The molecular ion is seen as a small peak at m/z = 142.•Notice the series ions detected that correspond to fragments that differ by 14 mass units, formed by the cleave of bonds at successive -CH2- units
REFRENCES
Chaintreau A. Simultaneous Distillation–Extraction: From Birth to Maturity – Review Flavour and Fragrance Journal 2001; 16(2) 136-148.
Flotron V, Houessou JK, Bosio A, Delteil C, Bermond A, Camel V. Rapid Determination of Polycyclic Aromatic Hydrocarbons in Sewage Sludges Using Microwave-Assisted Solvent Extraction. Comparison with Other Extraction Methods. Journal of Chromatography. A 2003; 999(1-2) 175-84.
Rice SL, Mitra S. Microwave-Assisted Solvent Extraction of Solid Matrices and Subsequent Detection of Pharmaceuticals and Personal Care Products (Ppcps) Using Gas Chromatography–Mass Spectrometry. Analitica Chimica Acta 2007; 589 125-132.
Hubschmann HJ. Handbook of GC-MS: Fundamentals and Applications. 2d Ed. Weinheim: Wiley-VCH; 2009.
Laskin J, Lifshitz C. Principles of Mass Spectrometry Applied to Biomolecules. New York: John Wiley and Sons; 2006.