Gas chromatography

InstrumentationComponents of a gas chromatograph

• The components include the– Mobile phase (Carrier gas) supply and pressure

and flow rate regulators– Injector – the column – the detector – The read out

Schematic of a typical Gas chromatograph

An example of the GC separation

Schematic Diagram of a Gas Chromatograph

Gas Chromatography System

Filters/Traps

Air

Hydrogen

Gas C

arrier

Column

• gas system

• inlet• column• detector• data

system

Data system

Syringe/Sampler

Inlets

Detectors

Regulators

H

RESET

Gas Flow

Main components of a typical gas chromatograph

– The mobile phase that transports the analytes through the column is a gas and is referred to as the carrier gas.

– The carrier gas flow, which is precisely controlled, allows great precision in the retention times.

– The analysis starts when a small quantity of sample in liquid orgaseous state is injected.

– The dual role of the injector is to vaporize the analytes and to mix them uniformly in the mobile phase.

– Once the sample is vaporized in the mobile phase, it is swept into the column,

– The column is usually a tube coiled into a very small section with a length that can vary from 1 to over 100 m.

– The column containing the stationary phase is situated in a variable temperature oven.

– At the end of the column, the mobile phase passes through a detector before it exits to the atmosphere.

Carrier Gas• Carrier gases, must be chemically inert, • Include helium, argon, nitrogen, carbon dioxide, and

hydrogen. • The choice of gases is often dictated by the detector

used. • Associated with the gas supply are pressure

regulators, gauges, and flow meters.

• The carrier gas system often contains a molecular sieve to remove water or other impurities.

• Detector gases - none or air/H2 (Flame ionization detector)

Carrier gas and flow regulation

• mobile phase is a gas (helium, nitrogen or hydrogen) • Flow rate of the carrier gas is 1 to 25 ml/min

depending on column type.• The carrier gas should not contain traces of water or

oxygen because these cause deterioration of the stationary phase.

• A filtering system containing a drying agent and a reducing agent is used between the gas source and the chromatograph.

• In gas chromatography, the nature of the carrier gas does not significantly alter the partition coefficient K between the stationary and mobile phases.

• However, the viscosity of the carrier gas and its flow rate have an effect on the analyte dispersion in the column (cf. van Deemter equation) thus affecting the efficiency and sensitivity of detection

• The pressure at the head of the column (several tens to hundreds of kPa) is stabilized either mechanically or through the use of an electronic device ensuring that flow rate (linear velocity of the gas) in the column is at its optimal value.

• When a temperature program is used during analysis, the viscosity of the mobile phase is increased thus increasing the resistance to carrier gas flow. It is, therefore, desirable to correct the pressure to compensate for this effect.

Optimum linear velocity and viscosity of carrier gas.

Sample introduction and the injection chamberSample introduction

• The sample, usually in the form of a solution and a typical injection size is 0.5µL

• Sample is introduced into the chromatograph with a microsyringe• Several types of syringe exist because of the diversity of injectors and

columns. • For gaseous samples, loop injectors can be used • In order to automate the injection and improve reproducibility,

manufacturers provide autosamplers in which the syringe and injection procedure are totally automated.

• These autosamplers, which can handle several samples, are very reliable.

• They operate in a cyclic fashion, taking the sample, injecting it rapidly (0.2 s) and rinsing the syringe.

• The latter is very important to avoid cross-contamination of successive samples that have similar composition.

Typical 10 µL syringe used in GC

Gas Sampling Loops/Valves

• Valves give better reproducibility• Require less skill• Can be easily automated

Injection valve

Injectors

• Role of injectors– Works as an inlet for the sample– It vaporises and mix the sample with the carrier

gas before the sample enters the head of the column.

• The injection is of a value to the quality of the separation.

• The modes of injection and the characteristics of injectors vary depending on the type of column used in the analysis.

Direct vaporization injection• For packed columns and mega bore columns of 530

µm, which typically use a flow rate of 10 ml/min, direct vaporization is a simple way to introduce the sample.

• This type of injectors uses a metal tube with a glass sleeve or insert.

• The glass insert is swept by the carrier gas and heated to the vaporization temperature for the analytes undergoing chromatography.

• One end of the injector contains a septum made of silicone rubber that allows the syringe needle to pass through it into the system.

• The other end of the injector is connected to the head of the column

• The entire sample is injected into the column in a few seconds.

Direct vaporization injector used for packed columns

• Rubber septum serves for about 30 injections in ordinary care

• 5-10 injections in case of large syringes

Split/splitless injection• When capillary columns are used with small flow

rates, even the smallest of injection volumes can saturate the column.

• Injectors that can operate in two modes, with or without flow splitting, are used (called split/splitless).

• In the split mode, the carrier gas arrives in the vaporization chamber with a relatively large flow

• A vent valve separates the carrier gas flow into two parts of which the smallest enters the column.

• A device is used to regulate the vent rate (generally between 50 and 100 ml/min).

• The split ratio varies between 1 : 20 and 1 : 500.

split/splitless injector(the split is regulatedby valve 2). The exitlabelled 1 is called theseptum purge.

cold on-column injector

Split/splitless Injector

Cold On- Column Injection• It is used for samples (biological) that decompose,

rearrange, or be adsorbed if they contact the heated metal surface of the port

• It also helps minimizing the discrimination against compounds of different volatilities

• The needle extends directly into the column (the end of the needle penetrates the packing; this may cause damage to the needle)

• Packing length is adjusted so that the end of the needle is either or just ahead of the glass wool plug

Injection Port Temperature• The temp. should be 20-30 oC hotter than the boiling

point of the least volatile component– But low enough to prevent sample decomposition and

septum bleed

• Temp. may be checked by raising it and watching : Position, or area, or shape of the peaks. Drastic changes mean the temp. setting is high

• It should be 10% above that of the column to ensure rapid volatilization of the sample. The efficiency of the column is almost constant under this condition

• Components may be vaporized at a temp. ~100oC below its atmospheric boiling point

• Very high boiling point or temp. sensitive material can be handled by dilution with volatile solvent that permits lowering the injection temp.

This will lower the sensitivity!

• Try various temperatures until peak broadening becomes apparent

• With temp. programming techniques low injection temps become very practical. No rush to vaporize the high boiling components

Effect of injection port temperature on resolution

a: Methanol; b: Ethanol; c: ispropanolBoling points: 65 – 82 oC

Columns• Three types of columns can be used in gas

chromatography: packed columns, capillary columns and wide bore or `530' columns (which have a 530 ftm inner diameter)

• For packed columns, the stationary phase is deposited onto a porous support. For the latter two, the stationary phase is deposited onto or bound to the inner surface of the column.

Capillary columnCapillary column

Typical dimensions of OTC for GC

Al-clad fused silica GC column

Column Material

• Stainless Steel : most common.adsorbs some Compounds particularly polar ones & especially water.

• Copper tubing reacts with : amines, acetylenes, terpens & steroidsWidely used. It is good for trace water analysis.

– Copper oxide coating is reactive and can interfere in gas analysis.

– O2 must be excluded from the carrier gas

• Al is used but troublemaker due to reactive Al-oxide formation

• Plastics: are limited due to permeability & temperature limit (used for reactive or highly corrosive chemicals H2S, HF

• Teflon, polypropylene and nylon tubing are available.

• Glass: If glass were not difficult to form into columns & relatively fragile it would be the very best choice for tubing ( used for pesticides & steroid).

Packed Columns

• These columns are made of stainless steel or glass. • They have diameters of 1/8 or 1/4 in (3.18 or 6.35

mm) and range in length from 1 to 3 m. • The internal surface of the tube is treated to avoid

catalytic interactions with the sample.• These columns use a carrier gas flow rate of

typically 10 to 40 ml/min. • Although they are still used in approximately 10% of

cases for routine GC work, packed columns are not well adapted to trace analyses.

• Packed columns contain an inert and stable porous support on which the stationary phase can be impregnated or bound (varying between 3 to 25%).

• The solid support is made of spheres of approximately 0.2 mm in diameter, obtained from diatomites, silicate fossils (such as kieselguhr, tripoli) whose skeleton is chemically comparable to amorphous silica.

• These materials, which have a specific surface area ranging from 2 to 8 m2/g, have been commercialized by several companies such as Johns Manville, under the name of Chromosorb©, and are used universally.

• Other synthetic materials have been developed such as Spherosil®, made of small silica beads.

• All of these supports have a chemical reactivity comparable to silica gel because of the presence of silanol groups.

Adsorption on Column Packings(or Capillary Walls)

• Polar analyte species such as, Alcoholsor Aromatic hydrocarbons are adsorbed physically on the silicate surfaces.

• Adsorption results in distorted peaksbroadened with tail

• This catalytic activity may lead to sample decomposition

Reasons for adsorption activity

• Silicates + Water Silanolgroups on the silicate surface

SiO

OHOH

SiO

OH

SiO

OH

Si

• Si-OH groups have strong affinity for polar organic molecules

Treatment of Solid Supports

• Non-acid washed (NAW) – an untreated form• Acid washed (AW) – use HCl – Removes metals,

impurities, Reduces surface activity and absorption

• Acid washed – Dimethyldichlorosilane treated (AW-DMCS)

Si

OH

+ (CH3)2SiCl2

HClSiOSi CH3

CH3OH HCl

Cl

CH3

CH3

SiOSiCH3

CH3

Capillary columns

• are usually prepared from high purity fused silica obtained by the combustion of SiH4 (or SiC14) in an oxygen-rich atmosphere.

• The internal diameter varies from 0.1 to 0.35 mm and the length from 15 to 100 m.

• Capillary columns are usually coated on the outside with polyimide or a thin aluminium film.

• Polyimide mechanically and chemically protects the column (Tmax = 370 °C).

• The columns are coiled around a lightweight, metallic support.

• The internal surface of the silica is usually treated or silanized, depending on the technique used to bond the stationary phase.

The `530 µm' or wide bore column

• The column is made from a silica tube of 0.53 mm internal diameter with length varying from 5 to 50 m.

• These columns maintain the features of capillary columns.• These columns appeared fairly recently (1983) following

developments in the area of flexible silica tubing. • Depending on the supplier, they are also called Megabore©,

Macrobore© or Ultrabore©. • The flow rates used in these columns can be as high as 15 ml/min,

close to that used in packed columns. • Thus it is possible to replace a packed column by a 530 µm column

while retaining the same injector and detector. • The advantage of wide bore columns over packed columns is their

lack of bleeding (loss of stationary phase with time).

Characteristics of Stationary Liquid Phase

• The stationary phase should provide separation of the sample with a reasonable column life

• Suitable phase is chosen on the basis of :– Experience or Experiment.– It is desirable to have maximum information

about the sample composition : bp.range, components expected & their structure

• Stationary phases should have similar chemical structure to the sample components

α(α is the separation factor)

Criteria for Liquid Phases

1. Maximize differential solubility

2. High absolute solubility for sample (measured as tR)

• Solubility good but not too good.• Gas phase is inert & separation occurs only

in Liquid Phase.3. Thermal stability (Temp. Limitations)

(Maximum & Minimum temp.)

Above which degradationoccurs

Below which equilibrium isToo slow to occur

4. Chemical inertness towards ample components at temp. of operation

5. Strong attachment to the Solid Support.6. Low vapor pressure at the temp. Used

(otherwise it will bleed off the column).7. Reproducibility, availability, cost.

– Same liquid phase produces same results when bought from any source or from same source

Commonly Used Liquid Phases

PHASE TEM. LIMITS Good for

1. SQUALANE 0/125oC Nonpolar2. OV-1, SE-30 100/350 oC3. DEXSIL-300 50/350 Oc

(Most thermally stable)

4. OV-17; SP-2250 0-350 Moderately polar5. QF-1; OV-210; 0-275

SP-2401

6. CARBOWAX-20M 60/225 Strongle polar7. DEGS 20/2008. OV-275 20/250

Polysiloxanes

Polyethylene glycol

Stationary phases (solid type)

• These phases are composed of adsorbing materials: molecular sieves, alumina, porous glass and gels (such as Chromosorb© 100, Porapak® and PoraPLOT®), and graphitized carbon black.

• They are mainly used to separate gases or volatile compounds.

• Capillary columns made by deposition of these materials in the form of very fine particulates are called PLOT (porous layer open tubular) columns.

Column Temperature Effect (Isothermal Analysis)

Isothermal Chromatographic Analysis Is One Which Is Performed At A Constant Colum Temperature.

Conclusion

• Higher temp. enables rapid analysis but loss in resolution.

• Lower temp. achieves better resolution but longer analysis Time

Narrow Boiling Range Samples

• Isothermal column temperature should be used.

• Select temperature 20-50oC lower than boiling range of sample when thin films are handled.

• Use highest temperature that still allows adequate resolution and stability to shorten analysis time.

Conclusion:1. Better resolution of earlier peaks2. Latter peaks elute more rapidly3. Peak shapes are more uniform

Features of Detectors

• A device that measures physical properties (preferred), not chemical properties

• The detector generates an electrical signal proportional to the sample concentration

• Detector and connections must be hot enough (20 to 30 oC above the column temp. or the boiling point of the highest boiling component) so that condensation of the sample or liquid phase does not occur

• Peak broadening or disappearance is characteristic for condensation in the connections

• Ionization type detectors must be maintained at temp. high enough to avoid not only condensation of sample but also the water or by-products formed in thr ionization process

Most Common GC DetectorsMost common detectors roughly in order from most common

•Thermal Conductivity Detector (TCD or hot wire detector), •Flame Ionization Detector (FID), •Electron Capture Detector (ECD), •Photo Ionization Detector (PID), •Flame Photometric Detector (FPD), •Thermionic Detector •VERY expensive choices: Atomic Emission Detector (AED)

•Ozone- or Fluorine-Induced Chemiluminescence Detectors. •All of these (except the AED) produce an electrical signal that varies with the amount of analyte exiting the chromatographic column.• Fourier Transform Infrared Detector (FTIR)• Mass Spectrometer (MS)• Other: UV, FT-NMR

Schematic of a thermal conductivity detector, TCD

• Two pairs of TCDs are used in gas chromatographs. • One pair is placed in the column effluent to detect

the separated components as they leave the column.• Another pair is placed before the injector or in a

separate reference column. • The resistances of the two sets of pairs are then

arranged in a bridge circuit. • The heated element may be a fine platinum, gold, or

tungsten wire or, alternatively, a semi conducting thermistor.

• The resistance of the wire or thermistor gives a measure of the thermal conductivity of the gas.

• Elution heat loss increased resistance needed to balance bridge = recorded

Flame Ionization Detector

Basic Principle• 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 compound is measured which is proportional to the carbon content of the molecule entering.

•sample burned in H2/air flame•sample must be combustible

•must use electrometer•ppm sensitivity•destructive

(FID)

Flame jet servesAs –Ve Electrode

Ring electrode: stainlessSteel gauze (+ve electrode)

Flame ionization Detector, FID Nitrogen phosphorus

detector, NPD

Thermionic DetectorNitrogen Phosphorus Detector (NPD)

• A special type of FID is called an alkali flame(AFID). Rubidium sulfate is burnet in the flame and the detector becomes specific for N and PN and P.

• Organics are not detected. Used for amines and nitrosoamines. (more commonly called the NPD)

View of FID

• The FID is a useful general detector for the analysis of organic compounds; – it has high sensitivity, – a large linear response range, – low noise. – robust and easy to use– unfortunately, it *destroys the sample.

Electron Capture DetectorElectron Capture Detector

Basic Principles of ECD• Radioactive source of 3H or 63Ni emits β particles.

• Ionization : N2 (Nitrogen carrier gas) + β (e) = N2+ + 2e (slow

electrons are formed)

• The slow electrons produce a steady background current “ a base line”

•X (F, Cl and Br) containing sample + (e- from the baseline current)) X-

Ion recombination : X- + N2+ = X + N2

•Thus, “base line” will decrease and this decrease constitutes the signal.

• Compounds detected: alkyl halides, conjugated carbonyles, nitriles, nitrates, organometallic compounds, Insecticides, pesticides, vinyl chloride, and fluorocarbons

Flame Photometric Detector, FPD

How does FPDfunction?

Column effluent + O2 Excited organic fragments

Combusted while

Surrounded by H2 envelopEm

itted radiationat certain w

avelength

Photomultiplierdetector

P at 510 and 526 nmS at 394 nm

Qualitative analysis by Chromatographic methods

• Qualitative analysis is based on retention data• Retention time tR is characteristic of a

substance, compared to a standard.• Reproducibility of retention depends upon

several experimental conditions:– column length and diameter, – stationary and mobile phases, – column packing,– column temperature, – mobile phase flow rate – and others

Retention time, tR

• tR: It is the time elapsed from the point of injection to the peak maximum

• Adjusted t’R : It is the time from the maximum of unretained peak (the peak of the mobile phase or the air peak) to the peak maximum of a certain component

• tM (hold up time): is the time required for the mobile phase to be eluted completely from the column

Unknown alcoholcomponents

Standard sample

Same column underSame conditionsHas been used

• Component 1 is used as the reference; it should be present or added to the sample and compatible with the sample

• Peak of component 1 must be close (but resolved) to the sample peak

When component 3 is suspected, add more of this component to the add more of this component to the sample and watchsample and watch any change in its peak

Basis for Quantitative Analysis• The peaks in the chromatogram are the basis for

quantitative analysisPeaks of interest should fulfill the following requirements:• must be undistorted• must be well separated• Must have a large S/N ratio • must have a flat baseline Peak shape: The ideal chromatographic peak is

symmetric and narrow

• Peak integrationThe peak height or, better, the area must be determined and this is done by the computer

• Calculation– External standard method– Internal standard method– Internal normalization

External standard method• This method is common to most quantitative analysis techniques. • It allows the measurement of the concentration of one or more

components that elute in a chromatogram containing, perhaps, many peaks.

• This method, employing the absolute response factor, K, is used in the following way (Single point calibration method)

Multilevel calibration

• In a multilevel calibration several different amounts of the standard are prepared and analyzed.

• A regression method is used (e.g. linear least-square) and this leads to a more precise value for Cunk.

• This quantitative method is the only one adapted to gaseous samples.

• This simple method is used in industry for repetitive analyses.

• For such analyses, chromatograph must be equipped with an autosampler,

Multilevel External Calibration of Fatty Acids

C

C

CDetector Response

Retention Time

14

16

18Peak Area (cm )

Sample Concentration (mg/ml)

2

4

6

8

10

0.5 1.0 1.5 2.0 2.5 3.0

2

T h e co n ten t % o f C fa tty ac id s =C

C + C + C 100∗14

181614

= th e co n ten t % o f C fa tty ac id s1 4

1 4

Internal standard method

• The areas of the compounds quantified are compared to the area of a reference compound, called an internal hard, present at a given concentration in each one of the samples

• This approach can compensate for imprecision due to the injected volume and instability between successive injections.

• In the following example, it is assumed that two compounds, 1 and 2, are to be qantifiedand that the internal standard is designated by IS.

Example illustrating the internal standard method.

Calculation of relative response factors

• A solution containing compound 1 at known concentration C1, compound 2 at concentration C2 and the internal standard at known concentration CISis prepared.

• Assuming that A1, A2and AlS represent the areas of the elution peaks.

• The quantities of each compound are m1, m2 and MIS.

• Given that mi = Ki Ai; thus– m1 = K1 A1; m2 = K2 A2; m3 = K3 A3

• Relative response factor can be derived as follows:

Calculation example

• The second step in the analysis is to obtain a chromatogram for a given volume of sample to which has been added a known quantity of compound IS, the internal standard.

• Assuming that A1’, A2’ and AIS' are the areas in the new chromatogram, and that m1’, m2’ and MIS’ are the quantities of compounds 1, 2 and IS introduced into the column under the same experimental conditions, then:

• The internal standard should have the following characteristics:

• It must be pure and not present initially in the sample• Its elution peak must be well resolved from the other

components of the sample• its retention time must be close to that of the compounds that

are to be quantified• Its concentration must be close to or above that of the

compounds that are to be quantified in order to obtain a linear response

• It has to be inert with respect to the sample components.

Internal Normalization

• The method of internal normalization (normalized to 100%) is used for mixtures in which each compound has been identified by its elution peak.

• Each of the peaks must be well separated from the others in order to fully characterize the sample.

• Assuming that the concentrations of three components (1, 2 and 3) present in a mixture are to be determined, the analysis is again carried out in two steps.

Internal normalization method

Calculation of relative response factor

• A reference solution containing the three compounds at known concentrations C1, C2 and C3 is prepared.

• The chromatogram obtained by injection of a volume V shows threepeaks of areas A1, A2 and A3.

• These areas will be related to the quantities m1, m2 and m3 of the compounds contained in the injected solution

• In this approach, one of the substances is considered for internal normalization.

• For example, if compound 3 is used to determine the relative response factors K1/3 and K2/3 of compounds 1 and 2 with respect to 3, one obtains:

Chromatography of the sample - calculation of concentrations

• A mixture containing components 1, 2 and 3 is analyzed by injection into the chromatograph.

• For the eluting peaks with areas A1’, A2

’ and A3

’, the %composition of the mixture represented as x1, x2 and x3 can be obtained from an expression of the following form: