Gas Chromatography

Chromatography is a group of methods to separate closely related components of complex mixture the sample is transported in a ‘mobile phase’ (gas or liquid), and passed through an immiscible ‘stationary phase’(column or solid surface) components of the sample distributes themselves between the mobile and the stationary phase to varying degree components that are strongly ‘retained’ by the stationary phase move slowly with the flow of mobile phase

as a result of these differences in mobility, sample components separate into discrete ‘bands’

Classification of Chromatographic Methods: 2 ways 1)based upon the physical means by which the stationary and mobile phases are brought into contact a)Column chromatography: stationary phase is held in a narrow tube through which the mobile phase is forced under pressure b)Planar chromatography: stationary phase is supported on a flat plate (TLC) or a paper (PC), and mobile phase moves by capillary action or under the influence of gravity

2) based upon the types of mobile and stationary phases and type of interaction of solutes

Gas Chromatography: the technique for the separation of thermally stable and volatile organic and inorganic compounds the sample is vaporized and injected onto the head of a chromatographic column elution is brought about by the flow of an inert gaseous mobile phase mobile phase mainly function to transport the analyte through the column Two types of GC: GSC and GLC

Instrumentation for GLC 1)Carrier gas supply - provides a constant flow of carrier (mobile phase) gas 2)Sample injection system - introduces sample vapors into the flowing gas stream 3)Column - contain stationary(liquid) phase 4)Oven - maintain the column at appropriate temperature 5)Detection and recording system - detect the sample components as they elute from the column

1)Carrier gas supply Carrier gases must be chemically inert and pure (helium, nitrogen, hydrogen) Choice of gases often dictate both column and detector performance Associated with the gas supply are pressure regulator, gauges and flow meter, to control flow rate, which is very important Many modern GC are equipped with electronic flow meters that are computer controlled to maintain the flow rate at any desired level

2) Sample injection System: most common method involves the use of a microsyringe to inject a liquid or gaseous sample through a self-sealing, rubber diaphragm or septum into a flash vaporizer port located at the head of the column in vaporizer the sample is vaporized and swept onto the column Flash vaporizer

Pyrolysis : t he technique of pyrolysis (controlled thermal fragmentation) has extended GC analysis to many low-volatility compounds (viz. rubber, polymer, paint films, resin etc) for ordinary columns,sample sizes vary from a few tenth of a µl to 20 µl for capillary column, sample size is much smaller 1nl –10nl, which is achieved by an injector-splitter

Derivative Formation: Derivatization prior to GC is often desirable to: improve the thermal stability of some compounds change the separation properties of some compounds introduce a detector-oriented tag into a molecule ex analysis of amino acids a) aminoacids are converted to butyl esters HCl by treating with 1-butanol and HCl (100 o C/30 min)

The butyl esters are converted into volatile N- trifluoroacetyl butyl esters Subsequent chromatographic resolution is then easily accomplished The trifluoro group, viz. trifluoroacetyl, pentafluoropropionly, and pentafluorobenzoyl, is commonly used for sensitizing substances to detection by electron capture

3) Columns: vary in length from 2m to 50m or more are constructed of stainless steel, glass, fused silica or Teflon in order to fit into an oven for thermostating they are usually coiled Two types of column are in general use; packed and open tubular or capillary column Packed columns are predominantly used till date

a) Packed Columns: are densely packed with a packing material (c/a solid support) coated with a layer of liquid stationary phase the ideal support consist of small uniform, spherical particles with good mechanical strength should be inert at elevated temperature most widely used support material is prepared from naturally occurring diatomaceous earth (skeletons of thousands of species of single-celled plants(diatoms))

Surface mineral impurities, which can serve as nonspecific adsorption sites, can be removed by acid washing Silanol (Si-OH) groups present on the surface of support causes peak tailing through H-bonding with polar samples This problem can be minimized by converting the silanol groups to silyl ether by treating the column packing with dimethyldichlorosilane Special packing material may be needed for particular application

b) Capillary or open tubular Columns In this the stationary phase is a uniform liquid film coating the interior of a capillary tubing provides separations much superior to packed columns in both speed and column efficiency : are of two basic types Wall-coated open tubular (WCOT)- are simply capillary tubes coated with a thin layer of the stationary phase

Support-coated open tubular (SCOT)- the inner surface of the capillary is lined with a thin film of support material (viz. diatomaceous earth)

The Stationary Phase: function of the stationary liquid phase is to separate the sample component into discrete peaks Desirable properties of the stationary phase include: a)low volatility- ideally, the boiling point of the liquid should be at 100 o C higher than the max operating temp for the column b)thermal stability c) chemical inertness proper choice is critical to success of a separation

4) Ovens: Chromatographic columns are coiled and held in a basket that is mounted inside an oven. The column oven must be able to be rapidly heated and cooled Ovens are usually constructed of low-mass stainless steel

Ideal detector for GC has following characteristics: Adequate sensitivity, good stability and reproducibility, linear response to solutes extending over orders of magnitude, good temperature range (RT o C), short response time, high reliability and ease of use, and nondestructive of sample Various types of detectors are available No detector exhibits all of these characteristics 5) Detectors: detect the sample components as they elute from the column

a) Flame Ionization Detector: Column effluent is mixed with H 2 and air and then ignited electrically Organic compounds produces ions and electrons conducting electricity through the flame

A potential is applied across the burner tip and collector electrode and resulting current is measured FID responds to the number of carbon atoms entering the detector per unit time The detector is insensitive towards moisture and certain gases (H 2 O, CO 2, SO 2 and NO x ) This makes it particularly useful for the detection of pollutants in natural water sample (contaminated with water and oxides)

b) Thermal Conductivity Detector: Based upon changes in the thermal conductivity of the gas stream due to presence of analyte molecule The sensing element is an electrically heated wire whose temperature depends upon the thermal conductivity of surrounding gas gas

The resistance of the wire gives a measure of the thermal conductivity of the gas Difference in thermal conductivity between the gas stream out of column (containing the separated analytes) and the reference stream of pure gas generate the signal Which is amplified to drive a recorder pen to a proportional height on a strip chart recorder Advantages: simplicity, nondestructive character, response to both organic and inorganic species

Interfacing GC with Spectroscopic methods : GC is often coupled with selective techniques of spectroscopy to form ‘ hyphenated methods’ provide very powerful tools for identifying the components of complex mixture a)Gas Chromatography/Mass Spectrometry (GC/MS) b)Gas Chromatography/Fourier Transform Infrared Spectroscopy (GC/FTIR)

GC equipment can be directly interfaced with rapid- scan mass spectrometers of various type Flow rate from capillary columns is low and the column output can be fed directly into the ionization chamber of the MS Gas Chromatography/Mass Spectrometry (GC/MS)

for packed columns, a jet separator is employed to remove most of the carrier gas from the analyte

simplest mass spectrometer for use in GC is the ‘ion trap mass spectrometer’ in this instrument, ions are created from the eluted sample by electron impact or chemical ionization ions are then ejected from the storage area to an electron multiplier detector so that scanning on the basis of mass-to-charge ratio is possible

GC/MS have been used for the identification of hundreds of components present in natural and biological systems characterization of the odor and flavor components of foods, identification of water pollutants, medical diagnosis based on breath components, and studies of drug metabolites

GC/FTIR provides a powerful means for separating and identifying the components of difficult mixture A narrow light pipe (cell) is connected to the GC column by means of narrow tubing light pipe is a glass tube with a gold coating on the inside and IR transmitting windows at the end IR beam travels down the light pipe by multiple reflection from the gold coating Gas Chromatography/Fourier Transform Infrared Spectroscopy (GC/FTIR)

Scanning is triggered by the output from a chromatographic peak detector The structural information and functional groups obtained from an IR spectrum provides valuable information about the compounds

Gas-Solid Chromatography: is based upon ‘adsorption’ of gaseous substances on solid surface is useful for the separation of species that are not retained by GLC (ex component of air, hydrogen sulfide, carbon disulfide, nitrogen oxide, CO, CO 2 ) GSC is performed with both packed and open tubular columns For the latter, a thin layer of the adsorbent is affixed to the inner walls of the capillary, such columns are c/a porous layer open tubular (PLOT) column

Two types of adsorbents are encountered: molecular sieves and porous polymers Molecular Sieves are aluminum silicate ion exchanger classified according to the max diameter of molecules that can enter the pores molecules smaller than the dimension of bead penetrate bead where it gets adsorbed

b) Porous Polymers: Are made from styrene cross-linked with divinylbenzene Pore size of the beads is uniform and controlled by the amount of crosslinking Widely used in separation of gaseous polar species

Summary: Carrier gas supply – Sample injection system – Column – Oven – Detection and recording system - Next time HPLC!!