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.