lecture 03 - gas chromatography .pptx

GAS CHROMATOGRAPHY

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Gas Chromatography Liquid Chromatography High Performance Liquid ChromatographyIon Chromatography

• History of gas chromatography• Principle• GC instrumentation • Carrier gas• Flow rate control• Types of injectors• Gas chromatography column• Column oven• Detectors• Headspace technique

Summary

History of Gas chromatography

Chromatography dates to 1903 in the work of the Russian scientist, Mikhail Semenovich Tswett.

German graduate student Fritz Prior developed solid state gas chromatography in 1947.

Archer John Porter Martin, who was awarded the Nobel Prize for his work in developing liquid-liquid (1941) and paper (1944) chromatography, laid the foundation for the development of gas chromatography and later produced liquid-gas chromatography (1950).

Principle

Gas chromatography - specifically gas-liquid chromatography - involves a sample being vapourised and injected onto the head of the chromatographic column.

The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.

The gaseous compounds being analyzed interact with the walls of the column, which is coated with different stationary phases.

This causes each compound to elute at a different time, known as the retention time of the compound.

The comparison of retention times is what gives GC its analytical usefulness.

Gas chromatography is in principle similar to column chromatography (as well as other forms of chromatography, such as HPLC, TLC), but has several notable differences.

Firstly, the process of separating the compounds in a mixture is carried out between a liquid stationary phase and a gas moving phase, whereas in column chromatography the stationary phase is a solid and the moving phase is a liquid.

Principle

(Hence the full name of the procedure is "Gas-liquid chromatography", referring to the mobile and stationary phases, respectively.)

Secondly, the column through which the gas phase passes is located in an oven where the temperature of the gas can be controlled, whereas column chromatography (typically) has no such temperature control.

Thirdly, the concentration of a compound in the gas phase is solely a function of the vapor pressure of the gas.

Principle

Gas chromatography is also similar to fractional distillation, since both processes separate the components of a mixture primarily based on boiling point (or vapor pressure) differences.

However, fractional distillation is typically used to separate components of a mixture on a large scale, whereas GC can be used on a much smaller scale (i.e. microscale).

Gas chromatography is also sometimes known as vapor-phase chromatography (VPC), or gas-liquid partition chromatography (GLPC).

Principle

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Principle

These alternative names, as well as their respective abbreviations, are frequently found in scientific literature. Strictly speaking, GLPC is the most correct terminology, and is thus preferred by many authors

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GC instrumentation

11

1

6

waste

1. Mobile phase bottle2. Online degasser3. M.P. derivaring pomp4. Sample injector5. Guard column

6. Separation column7. Column oven8. Detector9. HPLC work station

32 4 5 8

9

7

Configuration of Tentative HPLC system

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Configuration of Tentative GC system

1. Carrier gas2. Flow rate control part 3. Sample injector4. Separation column5. Column oven6. Detector7. GC work station1

3

5

4

6

7

2

Commercially available GC detectors and their applications

Detector Application

Flame Ionization (FID) Carbon compounds

Thermal Conductivity (TCD) Universal

Mass spectrometer (MSD) Variety of compounds

Electron capture (ECD) Halogenated compounds

Flame Photometric (FPD) S & P compounds

Atomic emission (AED) Metals, Halogen, C & O compounds

Radioactivity 3H & 14C compounds

Chemiluminescent Sulfur containing compounds

Photoionization (PID) Aromatic compounds

Nitrogen / Phosphorous (NPD) N,P & halogen compounds

Electroconductivity (ECD) S & N compounds

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The carrier gas must be chemically inert.

Commonly used gases include nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependant upon the type of detector which is used.

The carrier gas system also contains a molecular sieve to remove water and other impurities.

Carrier gas

Common gases--- Helium, Hydrogen, Nitrogen, argon

Requirements----- 1) Suitable for particular detector in use. 2) Inert

3) Dry and pure 4) Free from organic

impurities 5) Regulated

Carrier gas

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Flow rate control

While flow is relatively easy to control, it still must be measured.

Bubble meters - post column or detector measurement of flow. Cheap and relatively accurate.

Rotameters - pre-column measurement of flow via position of ball floating in a calibrated glass tube.

Electronic flow sensor - A modified thermal conductivity detector.Permits continuous measurement of flow over a reasonably large range. Must be calibrated for accurate flow measurement.

Response will also vary based on carrier gas used.

Types of injectors

1) Packed column injectors

2) Split / splitless injectors

3) On column Capillary injectors

4) Programmable split/splitless injectors

Split / splitless injector

For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a "plug" of vapour - slow injection of large samples causes band broadening and loss of resolution.

The most common injection method is where a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at the head of the column.

The temperature of the sample port is usually about 50°C higher than the boiling point of the least volatile component of the sample.

For packed columns, sample size ranges from tenths of a microliter up to 20 microliters.

Capillary columns, on the other hand, need much less sample, typically around 10-3 mL.

For capillary GC, split/splitless injection is used. Have a look at this diagram of a split/splitless injector;

The injector can be used in one of two modes; split or splitless.


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The injector contains a heated chamber containing a glass liner into which the sample is injected through the septum.

The carrier gas enters the chamber and can leave by three routes (when the injector is in split mode).

The sample vapourises to form a mixture of carrier gas, vapourised solvent and vapourised solutes.

A proportion of this mixture passes onto the column, but most exits through the split outlet. The septum purge outlet prevents septum bleed components from entering the column


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The choice of column depends on the sample and the active measured.

The main chemical attribute regarded when choosing a column is the polarity of the mixture, but functional groups can play a large part in column selection.

The polarity of the sample must closely match the polarity of the column stationary phase to increase resolution and separation while reducing run time.

The separation and run time also depends on the film thickness (of the stationary phase), the column diameter and the column length.

Gas chromatography column

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Gas chromatography columns are of two designs: packed or capillary.

Packed columns are typically a glass or stainless steel coil (typically 1-5 m total length and 5 mm inner diameter) that is filled with the stationary phase, or a packing coated with the stationary phase.

Capillary columns are a thin fused-silica (purified silicate glass) capillary (typically 10-100 m in length and 250 mm inner diameter) that has the stationary phase coated on the inner surface.


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Capillary columns provide much higher separation efficiency than packed columns but are more easily overloaded by too much sample.



Carrier gas Sample

Gas

Adsorption separation

Liquid

Solid

Distributive segregation


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Gas / solid Chromatography(A) Packed column (Adsorption Chromatography)

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Stationary phase

OH OH OH

Si O Si O Si

silica particle

Mobile phase


Synthetic zeolite Silica gel Activated carbon Activated alumina

Column packing material

Nitrogen Helium Argon Carbon dioxide

Carrier gas

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Stationary phase

Highly polar base material

OH OH OH

Si O Si O Si

silica particle

Mobile phase

Low polar base material

Hexane (C6H14)

Ethyl acetate (C4H8O2)

Chloroform (CHCl3)

(A) Normal phase separation column

Adsorption Chromatography

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http://ja.wikipedia.org/wiki/%E3%83%95%E3%82%A1%E3%82%A4%E3%83%AB:Chloroform_3D.svg

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OH OH OH

Si O Si O Si

silica particle

OH OH OH

Si O Si O SiO

C6H14

C6H14 C6H14

C6H14

C6H14

C6H14 C6H14C6H14

C6H14 C6H14

C6H14

C6H14 C6H14

C6H14

C6H14

C6H14

C6H14 C6H14

C6H14C6H14

Stationary phase: High polarity

Mobile phase : Low polarity

1. Ethylene glycol2. Glycerine

3. Erythritol

4. Xylitol

5. Mannitol

6. Inositol

High polarity analytes



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1. Ethylene glycol (C2H4(OH)2)2. Glycerine (C3H5(OH)3)

3. Erythritol (C4H6(HO)4)

4. Xylitol (C5H7(OH)5)

5. Mannitol (C6H8(OH)6)

6. Inositol (C6H6(OH)6)

Low polarity

High polarity

Retention time / min



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OH OH OH

Si O Si O Si

silica particle

OH OH OH

Si O Si O SiO

N2

N2 N2N2

N2N2 N2

N2

N2 N2

N2

N2

N2

N2

N2

N2

N2 N2

N2N2


Mobile phase : Nitrogen


N2

N2

N2

N2

N2N2

N2

N2

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1

2

3 45

6


1. Benzene2. Toluene3. Acetone4. Iso-Propanol5. Ethanol6. Methanol

Impurities in methanol

Low polarity

High polarity

Basic Separating Principles in HPLC : Polarity of Mobile phase

Mobile phase/solvent Polarity index (P’)

Cyclohexane 0.04

n-hexane 0.1

hexanol 0.6

carbon tetrachloride 1.6

propyl ether 2.4

toluene 2.4

diethyl ether 2.8

tetrahydrofuran 4.0

ethanol 4.3

ethyl acetate 4.4

dioxane 4.8

methanol 5.1

acetronitrile 5.8

water 10.2

Low polarity

High polarity

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R = CnH(2n+1)

About alcohol

n = 1 CH3OHn = 2 C2H5OHn = 3 C3H7OHn = 4 C4H9OHn = 5 C5H11OHn = 6 C6H13OHn = 7 C7H15OHn = 8 C8H17OHn = 9 C9H19OHn = 10 C10H21OH

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1

2

3

4 5

1. CO2

2. O2

3. N2

4. CH4

5. CO

Inert gas

In 1979, a new type of WCOT column was devised - the Fused Silica Open Tubular (FSOT) column; fused-silica wall-coated (FSWC) columnthe walls of the fused-silica columns are drawn from purified silica containing minimal metal oxides.

Much thinner than glass columns, with diameters as small as 0.1 mm and lengths as long as 100 m. Polyimide coating gives strength. flexible and can be wound into coils.

commercially available and currently replacing older columns due to increased chemical inertness, greater column efficiency and smaller sampling size requirements.


Capillary columns have an internal diameter of a few tenths of a millimeter.

Two types – Wall-coated open tubular (WCOT)

consist of a capillary tube whose walls are coated with liquid stationary phase.

– Support-coated open tubular (SCOT)inner wall of the capillary is lined with a thin layer

of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed.


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SCOT columns are generally less efficient than WCOT columns.

Both types of capillary column are more efficient than packed columns.


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Gas / Liquid Chromatography

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Stationary phase

Highly polar base material

OH OH OH

Si O Si O Si

silica particle

Mobile phase


(B) Fused silica open tubular (FSOT) column (Adsorption Chromatography)

Gas / solid Chromatography


Carrier gas

Fused silica gel




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OH OH OH

Si O Si O Si

OH OH OH

Si O Si O SiO

N2

N2 N2

N2

N2

N2 N2N2

N2 N2

N2

N2 N2

N2

N2

N2

N2 N2

N2N2



1. Ethylene glycol2. Glycerine

3. Erythritol

4. Xylitol

5. Mannitol

6. Inositol

High polarity analytes



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1

23

45

1. i-Propanol2. i-Butanol3. i-Amyl alcohol4. Ethyl acetate5. n-Propyl acetate6. n-Butyl acetate

Impurities in methanol

6

Gas / Liquid Chromatography


Carrier gas

(C) Fused-silica wall-coated (FSWC) column (partition chromatography)


Synthetic zeolite Silica gel Activated carbon Activated alumina

Dimethylpolysiloxane Polyethyleneglycol Diothyleneglycol succinate

O

N2

N2

N2

N2

N2

N2N2

N2

N2 N2

N2

N2

N2

N2

N2

N2

N2 N2

N2

N2

Stationary phase: Polyethyleneglycol


C

C H

H

OH

O

C

C H

H

OH

H

H

O

C

C H

H

OH

H

H

O

C

C H

H

OH

H

H

O

C

C H

H

OH

H

Hn n n nn

H H H H H

1. Acetone (CH3COCH3)2. Ethyl acetate (CH3COOC2H5) 3. Methyl ethyl ketone (C4H6(HO)4)

4. Methanol (CH3OH)

5. Isopropanol (CH3CH(OH)CH3)

6. Ethanol (C2H5OH)

7. Toluene (C7H8 (C6H5CH3))

Gas / Liquid Chromatography(C) Fused-silica wall-coated (FSWC) column (partition chromatography)

1. Acetone (CH3COCH3)2. Ethyl acetate (CH3COOC2H5) 3. Methyl ethyl ketone (C4H6(HO)4)

4. Methanol (CH3OH)

5. Isopropanol (CH3CH(OH)CH3)

6. Ethanol (C2H5OH)

7. Toluene (C7H8 (C6H5CH3))

1 2

3

4

56

7



1. Acetone2. 2-Butanol3. 1-Propanol4. 2-Methyl-1-propanol5. Allyl alcohol6. 1-Butanol7. 3-Methyl-1-butanol8. 1-Pentanol

1

23

45

67

8

Short Chain Alcohols


1. Methanol (CH3OH)2. Ethylens3. Ethane4. Propylene5. Propane6. Iso-Butane7. Iso-Butene8. n-Butane9. trans-2-Butene10.Cis-2-Butene11. iso-Pentane12.n-Pentane

1

23

45

6

7

8

9 10 11 12

Typical Gas Chromatogram

Courtesy of Aerosol Dynamics Inc., Berkeley, CA

Column oven

A gas chromatography oven, open to show a capillary column.

The column(s) in a GC are contained in an oven, the temperature of which is precisely controlled electronically.

When discussing the "temperature of the column," an analyst is technically referring to the temperature of the column oven.

The rate at which a sample passes through the column is directly proportional to the temperature of the column.

The higher the column temperature, the faster the sample moves through the column.

However, the faster a sample moves through the column, the less it interacts with the stationary phase, and the less the analytes are separated.

In general, the column temperature is selected to compromise between the length of the analysis and the level of separation.

Column oven

A method which holds the column at the same temperature for the entire analysis is called "isothermal."

Most methods, however, increase the column temperature during the analysis, the initial temperature, rate of temperature increase (the temperature "ramp") and final temperature is called the "temperature program.“

A temperature program allows analytes that elute early in the analysis to separate adequately, while shortening the time it takes for late-eluting analytes to pass through the column.

Column oven

There are many detectors which can be used in gas chromatography.

Different detectors will give different types of selectivity.

A non-selective detector responds to all compounds except the carrier gas, a selective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound.

Detectors can also be grouped into concentration dependant detectors and mass flow dependant detectors.

Detector

The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample Dilution of with make-up gas will lower the detectors response.

Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector.

The response of a mass flow dependant detector is unaffected by make-up gas.

Have a look at this tabular summary of common GC detectors:

Detector

General characteristic in choosing detectors:Sensitivity

– Measure of the response of the detector per unit of change of sample

– Able to detect low concentration doesn’t mean good sensitivity, its about the response towards change

– But limit of detection (LOD) commonly been used is 3×S/N ratio

Response and response time– Must show rapid response towards presence of

analyte

Detector

General characteristic in choosing detectors:Selectivity and specificity

– Selectivity: the difference of response of detector towards different compounds or group of compounds

– Specific: Discrimination towards analyte based on presence of a specific element or functional group

Linear response range– An ideal detector respose must be linear with the

quantity of solute to be determined graph of response vs must be a straight line

– range of sample amount been measured from the point of LOD to the point where the correlation reaches 5% deviation from the straight line

Detector

Commercially available GC detectors and their applications

Detector Application

Flame Ionization (FID) Carbon compounds

Thermal Conductivity (TCD) Universal

Mass spectrometer (MSD) Variety of compounds

Electron capture (ECD) Halogenated compounds

Flame Photometric (FPD) S & P compounds

Atomic emission (AED) Metals, Halogen, C & O compounds

Radioactivity 3H & 14C compounds

Chemiluminescent Sulfur containing compounds

Photoionization (PID) Aromatic compounds

Nitrogen / Phosphorous (NPD) N,P & halogen compounds

Electroconductivity (ECD) S & N compounds

The effluent from the column is mixed with hydrogen and air, and ignited.

Organic compounds burning in the flame produce ions and electrons which can conduct electricity through the flame.

A large electrical potential is applied at the burner tip, and a collector electrode is located above the flame.

The current resulting from the pyrolysis of any organic compounds is measured.

Flame ionization detector

FIDs are mass sensitive rather than concentration sensitive; this gives the advantage that changes in mobile phase flow rate do not affect the detector's response.

The FID is a useful general detector for the analysis of organic compounds; it has high sensitivity, a large linear response range, and low noise.

It is also robust and easy to use, but unfortunately, it destroys the sample

Highly sensitive to organic compound


Little or no response to water, carbondioxide, carrier gas impurities and hence zero signal when no sample is present

Stable baseline is not significantly affected by fluctuation of carrier gas, flow rate and pressure


Thermal Conductivity Detector (TCD)

Not as sensitive as others, but it is non-specific and non-destructive.

electrically-heated wire or thermostat.

The temperature of the sensing element depends on the thermal conductivity of the gas flowing around it.

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Changes in thermal conductivity, such as when organic molecules displace some of the carrier gas, cause a temperature rise in the element which is sensed as a change in resistance.

Thermal Conductivity Detector (TCD)

Photoionization detector (PID)

Selective determination of aromatic hydrocarbons or organo-heteroatom species. Uses ultraviolet light as a means of ionizing an analyte exiting from a GC column.

The ions produced by this process are collected by electrodes.

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Photoionization detector (PID)

The current generated is therefore a measure of the analyte concentration.

Electron capture detector (ECD)

Uses a radioactive Beta emitter (electrons) to ionize some of the carrier gas and produce a current between a biased pair of electrodes.

When organic molecules that contain electronegative functional groups (e.g.: halogens, phosphorous, and nitro groups) pass by the detector, they capture some of the electrons and reduce the current measured between the electrodes.

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As sensitive as the FID but has a limited dynamic range and finds its greatest application in analysis of halogenated compounds.

Electron capture detector (ECD)

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Mass spectrometer detector (MSD)

Mass spectrometry (MS) is an analytical technique that produces spectra (singular spectrum) of the masses of the molecules comprising a sample of material.

The spectra are used to determine the elemental composition of a sample, the masses of particles and of molecules, and to elucidate the chemical structures of molecules, such as peptides and other chemical compounds.

Mass spectrometry works by ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios.

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Mass spectrometer detector (MSD)

Heater

Source of electrons to bornbard sample

To vacuum pump

Work station

Electric field

Path of heavy ions

Detector

Path of lighter ions

Electromagnet to produce a variable magnetic field

Path of ion of right mass to be detected

Basic principles of headspace analysis

Simple Definition: The 'headspace' is the gas space in a chromatography vial above the sample. Headspace analysis is therefore the analysis of the components present in that gas.

Headspace GC is used for the analysis of volatiles and semi-volatile organics in solid, liquid and gas samples.

The popularity of this technique has grown over recent years and has now gained worldwide acceptance for analyses of alcohols in blood and residual solvents in pharmaceutical products

A headspace sample is normally prepared in a vial containing the

sample, the dilution solvent, a matrix modifier and the headspace.

Volatile components from complex sample mixtures can be

extracted from non-volatile sample components and isolated in

the headspace or gas portion of a sample vial.

A sample of the gas in the headspace is injected into a GC

system for separation of all of the volatile components.



Phases of the Headspace Vial

G = the gas phase (headspace)The gas phase is commonly referred to as the headspace and lies above the condensed sample phase.

S = the sample phaseThe sample phase contains the compound(s) of interest. It is usually in the form of a liquid or solid in combination with a dilution solvent or a matrix modifier.

Once the sample phase is introduced into the vial and the vial is sealed, volatile components diffuse into the gas phase until the headspace has reached a state of equilibrium as depicted by the arrows. The sample is then taken from the headspace


1) Place Sample in vial and seal it.

2) Place vial in Headspace Autosampler.

3) Collect Analysis results from data station

- Automatic thermosetting to equilibrium for volatiles between the phases.

- Automatic injection onto GC separation column of the gas-phase “extract”.


Standby

Pressure-balanced time-based sampling

Carrier gas enters through Valve 1 (V1) and is directed partly through the heated transfer line to the GC and partly to the heated needle, which is constantly flushed to avoid cross contamination.

The needle flush gas exits through the needle vent valve (V2).

The following slides present the unique system of pressure balanced sampling, developed by PerkinElmer.

Standby

Pressurization

Prior to injection, each vial is pressurized to a preset pressure with carrier gas to ensure that all injections are performed under the exact same conditions, regardless of different equilibrium pressures in different samples.


Standby

Pressurization

Sampling

During injection, the carrier gas supply and the needle purge are switched off.

Sample flows to the separation/analysis system from the pressurized headspace vial.

The injected volume is proportional to

the injection time.


Standby

Pressurization

Sampling

Standby

At the end of injection, the carrier gas and the needle purge are once more switched on and the injected sample volume driven through the analytical system.

The needle is retracted and the system goes into standby mode.


Calibration & standards

The most straight forward method for quantitative chromatographic analysis involves the preparation of a semi rise of standard solutions that approximate the composition of the unknown.

Chromatograms for the standards are then obtained and peak heights or areas are plotted as a function of concentration.

A plot of the data should yield a straight line passing through the origin, analyses are based on this plot.

Internal standard method

The highest precision for quantitative chromatography is obtained by use of internal standards

because the uncertainties introduced by sample injection are avoided.

In this procedure, a carefully measured quantity of an Internal standard substance is introduced into each standard and sample, and the ratio of analyte to internal standard peak areas (or heights) serve as the analytical parameter.

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For this method to be successful, it is necessary that the internal standard peak be well separated from the peaks of all the other components of the sample.

Internal standard method

Area normalization method

Another approach that avoids the uncertainties associated with sample injection is the area normalization method.

Complete elution of all components of the sample is required.

In the normalization method, the areas of all eluted peaks are computed,

after correcting these areas for differences in the detector response to different compound types, the concentration of the analyte is found from the ration of its area to the total area of all peaks.

• History of gas chromatography• Principle• GC instrumentation • Carrier gas• Flow rate control• Types of injectors• Gas chromatography column• Column oven• Detectors• Headspace technique

Summary

lecture 03 - gas chromatography .pptx

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