Chapter 26 & 27

Introduction to Chromatographic Separations and Gas Chromatography

The concept of Gas-Liquid Chromatography was first proposed in 1941 by Martin

and Synge, who jointly were responsible for the development of liquid-liquid partition

chromatography. More then a decade passed before the true advantages of Gas-liquid

chromatography were demonstrated experimentally. Three years later in 1955 the first

commercial apparatus for GLS appeared in the market. Ever since its first introduction,

the growth and improvement in techniques, instrumentation and application has been

phenomenal.

Chromatography is an analytical method that is widely used for the separation,

identification and determination of chemical components in a complex mixture. No other

separation method is as powerful and generally applicable as chromatography. There are

numerous chromatography techniques, however they have in common the use of a

stationary phase and mobile phase. Components of a mixture are carried through the

stationary phase by a flow of a gaseous or liquid mobile phase, separations being based

on the differences in migration rates among the sample components. In contrast to most

other types of chromatography the mobile phase does not interact with molecules of the

analyte, its only function is to transport the analyte through the column. There are two

types of Gas Chromatography, Gas Solid Chromatography (GSC) and Gas Liquid

Chromatography (GLC). This site is mainly devoted to Gas Liquid Chromatography

In gas chromatography (GC), 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. In contrast to most other types of chromatography, the mobile phase does

not interact with molecules of the analyte; its only function is to transport the analyte

through the column.

Gas liquid chromatography is based upon the partition of analyte between a gaseous

mobile phase and a liquid phase immobilized on the surface of an inert solid. The

concept of gas-liquid chromatography was first enunciated in 1941 by Martin and Synge.

Principles of Gas-Liquid Chromatography

Retention Volumes:To take into account the effects of pressure and temperature in gas chromatography, it

useful to use the retention volumes. The specific retention volume, Vg is defined as

follows:

where, J = the pressure drop factor, F = average flow rate, tr and tm = retention times W

= mass of stationary phase and Tc = column temperature in Kelvin.

Instruments for GC

The schematic of a gas chromatograph is shown below:

Carrier Gas Supply

Carrier gases, which 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. In

addition, the carrier gas system often contains a molecular sieve to remove water or other

impurities.

Flow rates are normally controlled by a two-stage pressure regulator at the gas cylinder

and some sort of pressure regulator or flow regulator mounted in the chromatograph.

Inlet pressures usually range from 10 to 50 psi, which lead to flow rates of 25 to 150

mL/min with packed columns and 1 to 25 mL/min for open-tubular columns. Flow rates

can be established using a soap-bubble meter.

Sample Injection System:Column efficiency requires that the sample be of

suitable size and be introduced as a plug of vapor; slow

injection of oversized samples causes band spreading

and poor resolution. The most common method of

injection involves the use of a micro-syringe to inject a

liquid or gaseous sample through a silicone rubber

diaphragm or septum into a flash vaporizer port located

at the head of the column. For ordinary analytical columns, sample sizes vary from a few

tenths of a microliter to 20microliters. Capillary columns require much smaller samples

(~0.001microliters)

Column Configurations and Column Ovens:Two general types of columns are encountered in gas chromatography, packed and open

tubular, or capillary. To date, the vast majority of gas chromatography has been carried

out on packed columns. Chromatographic columns vary in length from less than 2m to

50m or more. They are constructed of stainless steel, glass, fused silica, or Teflon. In

order to fit into and oven for thermostating, they are usually formed as coils having

diameters of 10 to 30cm.

Column temperature is an important variable that must be controlled to a few tenths of a

degree for precise work, so the column is ordinarily housed in a thermostatted oven.

The optimum column temperature depends upon the boiling point of the sample and the

degree of separation required. Roughly, a temperature equal to or slightly above the

average boiling point of a sample results in a reasonable elution time. For samples with a

broad boiling range, it is often desirable to employ temperature programming, whereby

the column temperature is increased either continuously or in steps as separation

proceeds. The figure below shows the improvement brought about by temperature

programming. In general, optimum resolution is associated with minimal temperature;

the cost of lowered temperature, however, is an increase in elution time and therefore the

time required to complete an analysis.

Detectors

Characteristics Of The Ideal Detector:

1. Adequate sensitivity.2. Good stability and reproducibility.3. A linear response to analyses that extends overseveral orders of magnitude.

4. A temperature range from room temperature to

at least 400degC.

5. A short response time that is independent of flow rate.

6. High reliability and ease of use.7. Similarity in response toward all analyses.8. Nondestructive of sample.

Flame Ionization Detector:The flame ionization detector (FID) is one of the most widely used and generally

applicable detectors for gas chromatography. The effluent from the column is mixed with

hydrogen and air and then ignited electrically. Most organic compounds, when

pyrolyzed at the temperature of a hydrogen/air flame, produce ions and electrons that can

conduct electricity through the flame. A potential of a few hundred volts is applied

across the burner tip and a collector electrode is located above the flame. The resulting

current is then directed into a high impedance operational amplifier for measurement.

Functional groups such as carbonyl, alcohol, halogen, and amine, yield fewer ions or

none at all in a flame. In addition, the detector is insensitive toward noncombustible

gases such as H2O, CO2, SO2, and NOx. These properties make the flame ionization

detector a most useful general detector for the analysis of most organic samples,

including those that are contaminated with water and oxides of nitrogen and sulfur. The

flame ionization detector exhibits a high sensitivity, large linear response range, and low

noise. It is generally rugged and easy to use. A disadvantage of this detector is that it

destroys the sample. A diagram of a typical FID is shown in below.

Thermal Conductivity Detector:A very early detector for gas chromatography is based upon

changes in the thermal conductivity of the gas stream

brought about by the presence of analyte molecules. This

device is sometimes called a katharometer. The sensing

element of a katharometer is an electrically heated element

whose temperature at constant electrical power depends

upon the thermal conductivity of the surrounding gas. The

heated element may be a fine platinum, gold or tungsten wire or, alternatively, a

semiconducting resistor. The advantage of the thermal conductivity detector is its

simplicity, its large linear range, its general response to both organic and inorganic

species, and its nondestructive character. A limitation of the katharometer is its relatively

low sensitivity.

Thermoionic Detector:The thermoionic detector (TID) is selective toward organic compounds containing

phosphorous and nitrogen. It is similar in structure to the flame detector.

Electron-Capture Detector:Electron-capture detector (ECD) operates in

much the same way as a proportional

counter for measurement of X-radiation.

Here the effluent from the column passes

over a beta-emitter, such as nickel-63 or

tritium (adsorbed on platinum or titanium

foil). An electron from the emitter causes ionization of the carrier gas (often nitrogen)

and the production of a burst of electrons. In the absence of organic species, a constant

standing current between a pair of electrodes results from this ionization process. The

current decreases however in the presence of those organic molecules that tend to capture

electrons. The electron-capture detector is selective in its response, being highly

sensitive toward molecules that contain electronegative functional groups such as

halogens, peroxides, quinones, and nitro groups. Electron-capture detectors are highly

sensitive and possesses the advantage of not altering the sample significantly. On the

other hand, their linear response range is usually limited to about two orders of

magnitude.

Atomic Emission Detector (AED):The newest commercially available gas-

chromatographic detector is based upon

atomic emission. In this device, the

eluent is introduced into a microwave-

energized helium plasma that is coupled to

a diode-array optical emission

spectrometer. The plasma is sufficiently energetic to atomize all of the elements in a

sample and to excite their characteristic atomic emission spectra.

GC COLUMNS AND STATIONARY PHASEPacked Columns:Present day packed columns are fabricated from glass, metal (stainless steel, copper,

aluminum), or Teflon tubes that typically have lengths of 2 to 3 m and inside diameters

of 2 to 4mm. These tubes are densely packed with a uniform, finely divided packing

material, or solid support, that is coated with a thin layer of stationary liquid phase. The

tubes are formed as coils having diameters of roughly 15cm.

Solid Support Materials:The solid support in a packed column serves to hold the stationary phase in place so that

as large a surface area as possible is exposed to the mobile phase.

The ideal support consists of small, uniform spherical particles with good mechanical

strength and a specific surface area of at least 1m/g. In addition, the material should be

inert at elevated temperatures and be uniformly wetted by the liquid phase.

Particle Size Supports:The efficiency of a gas-chromatographic column increases rapidly with decreasing

particle diameter of the packing. The pressure difference required to maintain a given

flow rate of carrier gas, however, varies inversely as the square of the particle diameter;

the latter relationship has placed lower limits on the size of particles employed in gas

chromatography, because it is not convenient to use pressure differences that are greater

than about 50psi.

Open Tubular Columns:Open tubular, or capillary, columns are of two basic types, namely, wall-coated open

tubular (WCOT) and support-coated open tubular(SCOT). WCOT columns are simply

capillary tubes coated with a thin layer of the stationary phase. In SCOT columns the

inner surface of the capillary is lined with a thin film of a support material, such as

diatomaceous earth. This type of column holds several times as much stationary phase as

does a wall-coated column and thus has a greater sample capacity.

ADSORPTION ON COLUMN PACKINGS OR CAPILLARY WALLSA problem that has plagued GC has been the physical adsorption of polar or polarizable

analyte species such as alcohols or aromatic hydrocarbons, on the silicate surfaces of

column packings or capillary walls. Adsorption results in distorted peaks, which are

broadened and often exhibit a tail. It has been established that adsorption is the

consequence of silanol groups that form on the surface of silicates by reaction with

moisture. The SiOH groups on the support surface have a strong affinity for polar

organic molecules and tend to retain them by adsorption. Support materials can be

deactivated by silanization with dimethyl chlorosilane (DMCS).

STATIONARY PHASE

Desirable properties for the immobilized liquid phase in a gas liquid chromatographic

column include:

1. low volatility2. thermal stability3. chemical inertness4. solvent characteristics such that k’ and alpha values forthe solutes to be resolved fall within a suitable range.

Generally, the polarity of the stationary phase should match that of the sample

components. Commonly used stationary phases are listed in below.

Stationary Phase Common

Trade Name

Maximum

Temperature (C)

Common Applications

Polydimethyl siloxane OV-1, SE-30 350 General-purpose nonpolar phase;

hydrocarbons; polynuclear aromatics;

drugs; steriods; PCBs

Poly(phenylmethyldimethyl)

siloxane (10% phenyl)

OV-3, SE-52 350 Fatty acid methyl esters; alkaloids;

drugs; halogenated compounds

Poly(phenylmethyl) siloxane

(50% phenyl)

OV-17 250 Drugs; steriods; pesticides; glycols

Poly(trifluoropropyldimethyl

) siloxane

OV-210 200 Chlorinated aromatics; nitroaromatics;

alkyl-substituted benzenes

Polyethylene glycol Carbowax 20M 250 Free acid; alcohol; ethers; essential oils;

glycols

Poly(dicyanoallyldimethyl)

siloxane

OV-275 240 Polyunsaturated fatty acids; rosin acids;

free acids; alcohols

Bonded and Cross-linked Stationary Phases:The purpose of bonding and cross-linking is to provide a longer-lasting stationary phase

that can be rinsed with a solvent when the film becomes contaminated. With use,

untreated columns slowly lose their stationary phase due to bleeding in which a small

amount of immobilized liquid is carried out of the column during the elution process.

Cross-linking is carried out in situ after a column is coated with one of the polymers

listed in above

Chiral Stationary Phases:There has been recent developments of stationary phases which can separate chiral

compounds. One method is to form a derivative of the analyte with an optically active

reagent that forms a pair of diasteromers that can be separated on an achiral column. The

alternative method is to use a chiral liquid as the stationary phase.

Film Thickness:Film thickness primarily affect the retentive character and the capacity of a column.

Thick films are used with highly volatile analytes, because such films retain solutes for a

longer time and thus provide a greater time for separation to take place. Thin films are

useful for separating species of low volatility in a reasonable time.

APPLICATIONS OF GC

Qualitative Analysis

Gas chromatographs are widely used as criteria for establishing the purity of organic

compounds. Contaminants, if present, are revealed by the appearance of additional

peaks.

The Retention Index:The retention index, I, was first proposed by Kovats in 1958 as a parameter for

identifying solutes from chromatograms. The retention index for any given solute can be

derived from a chromatogram of a mixture of that solute with at least two normal alkanes

having retention times that bracket that of the solute. That is, normal alkanes are the

standards upon which the retention index scale is based. By definition, the retention

index for a normal alkane is equal to 100 times the number of carbons in the compound

regardless of the column packing, the temperature, or other chromatographic conditions.

The retention index system has the advantage of being based upon readily available

reference materials that cover a wide boiling range.

Quantitative Analysis:The relative concentration of an analyte is proportional to the peak area obtained on the

gas chromatogram. Thus the GC can be calibrated with several standards and a

calibration curve is obtained, then the concentration of the unknown analyte can be

determined using the peak area.

INTERFACING GC WITH SPECTROSCOPIC METHODS

When GC is coupled with other separation methods, both serve as a powerful tool for

identifying the components of complex mixtures. A popular combination is GC/MS.

This interface have been used for the identification of hundreds of components that are

present in natural and biological systems. For example, these procedures have permitted

characterization of the odor and flavor components of foods, identification of water

pollutants, medical diagnosis based on breath components, and studies of drug

metabolites. Another popular interface is that of GC/FTIR. Below is a diagram of

GC/MS a instrument.

GAS-SOLID CHROMATOGRAPHY

Gas-solid chromatography is based upon absorption of gaseous substances on solid

surfaces. Distribution coefficients are generally much larger than those for gas-liquid

chromatography. Consequently, gas -solid chromatography is useful for the separation

of species that are not retained by gas-liquid columns, such as the components of air,

hydrogen sulfide, carbon disulfide, nitrogen oxides, and rare gases. Gas-solid

chromatography is performed with both packed and open tubular columns. For the latter,

a thin layer of adsorbent is affixed to the inner walls of the capillary. Such columns are

sometimes called porous layer open tubular columns, or PLOT columns. Two types of

adsorbents are molecular sieves and porous polymers.

Molecular Sieves:Molecular sieves are aluminum silicate ion exchangers, whose pore size depends upon

the kind of cation present. The sieves are classified according to the maximum diameter

of molecules that can enter the pores. Commercial molecular sieves come in pore sizes

of 4, 5, 10, and 13 angstroms. Molecular sieves can be used to separate small molecules

from large.

Porous Polymers:Porous polymer beads of uniform size are manufactured from styrene cross-linked with

divinylbenzene. The pore size of these beads is uniform and is controlled by the amount

of cross-linking. Porous polymers have found considerable use in the separation of

gaseous polar species such as hydrogen sulfide, oxides of nitrogen, water, carbon

dioxide, methanol, and vinyl chloride.

Important Terms to Know:

Stationary phase

The stationary phase consists of the support, which is either a porous small-particle material for packed columns or the inner walls of a glass or fussed-silica capillary column. The stationary liquid is either fixed merely by adhesion or by chemical bonding in order to produce a homogeneous coating.

Mobile phase.

In GC an inert gas e.g. He N2 H2 acts as the mobile phase, which effects the transport of the solutes through the column.

Elution

By transportation in the mobile phase the solutes migrate with a velocity through the separation column which depends on the partition coefficients of the solutes between the given stationary phase and mobile phase.

Solutes

Sample components that are being separated.

Detector

The detector generates a continuously recorded electrical signal during the chromatographic separation, which is proportional to the concentration of the separated solute in the mobile phase. There are universal and specific types of detectors.

Chromatography

A term for methods of separation based upon the partition of analyte species between a stationary phase and mobile phase.

Distribution constants, Partition coefficients

An equilibrium constant for the distribution of a solute between two immiscible phases.

Retention times

The time between sample injection and arrival of the analyte peak at the detector.

Gaussian Distribution

A distribution in which small departures from a central result occur more frequently then large ones and in which positive a negative variation occur with equal frequency.

GC Quiz Question

The following data were obtained by gas-liquid chromatography on a 40cm packed

column

Compound TR min W½ min

Air 1,9 -Methylcyclohexane 10.0 0.76Methylcyclohexene 1.09 0.82

Toluene 13.4 1.06Question 1

Calculate,1a) the average number of plates from the data1b) the standard deviation of the average in 1a1c) the average plate height for the column

Question 2

Calculate the resolution for,2a) Methylcyclohexene and Methylcyclohexane2b) Methylcyclohexene and toluene2c) Methylcyclohexane and toluene

Question 3

If Cs and Cm for the column were 19.6 and 62.6 mL, respectively and a non retained air

peak appeared after 1.9 min calculate the

3a) Retention factor for each of the three compounds3b) Distribution constant for each of the three compounds3c) Selectivity factor for methylcyclohexane and methylcyclohexene3d) Selectivity factor for methylcyclohexene and toluene

GC Quiz Answers

1a) N = 2.7 x 103

1b) s = 0.1 x 103 1c) H = 0.015cm2a) 1.12b) 2.72c) 3.73a) k’1 = 4.3 k’2 = 4.7 k’3 = 6.13b) K1 = 14 K2 = 15 K3 = 193c) 1.113d) 1.28

References:

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